Microbial Culture and Media Quiz

Bacterial Cultures

• Microbial culture is a method to grow microorganisms (bacteria and fungi) in a lab.
• In vitro cultures of bacteria allow for:
  • Isolation and cultivation of pathogenic bacteria
  • Morphology analysis of colonies and phenotypic characteristics for identification
  • Antibacterial susceptibility testing for choosing suitable antimicrobial agents
• Microbial culture identification is crucial to determine the causative agent of a disease.

Culture Media

• Culture media are nutrient media, artificial growth mediums for microbial propagation.
• Types of culture media are categorized by consistency (solid, semi-solid, liquid/broth).
• Categorized by nutritional components:
  • Complex media (undefined): contain unknown nutrients, used for fastidious bacteria (requiring extra nutrients like blood, serum, yeast or meat extract).
  • Synthetic media (chemically defined): contain known and well-defined chemical constituents.
• Categorized by function/property:
  • Selective media: promotes growth of certain bacteria while inhibiting others.
  • Differential media: allows differentiation of bacteria based on their metabolic activities (e.g. lactose fermentation).
  • Indicator media: show the presence of a specific substance in the medium.
  • Enriched media: support the growth of specific species that need extra nutrients.
  • Composite media: combination of properties.
  • Storage media: maintain viability of cultures.
  • Transport media: for safely carrying specimens for analysis.

Liquid Culture Media (Nutrient Broth)

• Nutrient Broth: liquid medium for nonfastidious microorganisms.
• Can be supplemented with blood, serum, sugars, etc. for special purposes.
• Typical formula (g/L): Beef Extract (1.0), Peptone (5.0), Yeast Extract (2.0), Sodium Chloride (5.0).
• Final pH 6.8 ± 0.2 at 25°C.
• Principle: components provide essential nutrients (amino acids, nitrogen, carbon, vitamins, minerals) and maintain osmotic balance for growth.

Solid Culture Media (Agar)

• Agar: unique solidifying agent from red-purple marine algae.
• Properties:
  • Stable during sterilization
  • Physiologically stable upon usage and storage
  • Nontoxic to bacterial growth.
  • Liquifies at 100°C, solidifies at 50°C
• Crucial for growing bacteria in agar plates for specific tests and observation.

Solid Medium for Bacteria (Nutrient Agar)

• Nutrient Agar ISO 6579: a non-selective medium for cultivating various microorganisms.
• Typical formula (g/L): Meat Extract (3.0), Peptone (5.0), Agar (15.0).
• Final pH 7.0 ± 0.2 at 25°C.
• Principle: Components supply essential nutrients (amino acids, nitrogen, carbon, minerals, vitamins) for microbial growth. Agar is the solidifying agent.

Microbial Growth Curve

• Stages: Lag phase, Log phase (exponential growth), Stationary phase, and Decline phase.

Continuous Culture Device (Chemostat)

• Chemostat: maintains a constant Log phase of bacterial growth.
• Flow control of fresh medium and outflow of spent medium.

Bacterial Growth vs Oxygen Concentration

• Obligate aerobes: require oxygen
• Obligate anaerobes: cannot tolerate oxygen
• Facultative anaerobes: can grow with or without oxygen
• Aerotolerant anaerobes: tolerate oxygen but do not use it for growth.

Bacterial Growth in Solid Media

• Observed as colony formation.
• Isolated colonies grow from single bacterial cells.
• Colony characteristics (e.g., shape, elevation, margin) aid in bacterial identification.

Bacterial Colony Morphology

• Categorized by form, elevation, and margin shape

Enriched Nonselective Media

• Blood agar: contains 5% sheep/horse blood to test hemolysis.

• Chocolate agar: modified blood agar via heating blood to a chocolate-brown colour.

• Mueller-Hinton agar: recommended for antibiotic susceptibility testing.

Disk Diffusion Test (Kirby-Bauer Test)

• Antimicrobial susceptibility test performed on Mueller-Hinton agar.
• Antibiotic discs are placed on the agar plate containing the microorganisms.
• Inhibition zones are measured to determine the effectiveness of different antibiotics.

Antibiotic Susceptibility Testing in Liquid Medium (MIC)

• Serial 2-fold dilutions of antibiotics in Mueller-Hinton broth.
• Inoculate with a known amount of microorganisms.
• Determine the lowest antibiotic concentration that inhibits bacterial growth (MIC).

Selective Media and Differential Media

• Selective media: specifically designed to recover particular bacteria from a mixture.
• Examples: Mannitol salt agar, Lowenstein-Jensen agar, CHROMagar.
• Selective media can also be differential to visually identify and differentiate.