Cultural Methods

• Culture media are substances that provide nutritional and environmental requirements for bacterial growth
• A culture is a group of microorganisms obtained in culture media
• Types of culture:

  • Pure culture: one species
  • Mixed culture: made up of organisms belonging to different species
  • Stock culture: pure culture of organisms used in supplying industry, research, etc
• Classification of culture media:

  1. According to consistency or physical state:

  • Agar: polysaccharides extracts of seaweed used as a base medium
  • Liquid culture medium: contains no agar, examples include Broth and Thioglycollate media
  • Semi-solid medium: contains gelatin or agar (0.5-1% of agar), example: SIM
  • Solid culture medium: pure agar (2-5% of agar), examples include BAP (Blood agar plate) and CAP (Chocolate agar plate)
• Classification of culture media:

  2. According to composition:

  • Synthetic culture medium: known ingredients, examples include Commercial media
  • Non-synthetic culture medium: composition is not known, examples include Ordinary broth
  • Tissue culture medium: made up of living cells
• Classification of culture media:

  3. According to how the medium is dispersed:

  • Plated medium: distributed in a petri dish (Richard Petri)
  • Tubed medium: distributed in a test tube, types include Slant, Butt, and Slant/butt
• Classification of culture media:

  4. According to use:

  • Simple medium: supports only the growth and multiplication of microorganisms, used for routine cultivation and maintenance, example: Nutrient broth
  • Enrichment medium: enhances propagation of certain organisms, examples include Selenite broth and Tetrathionate
  • Enriched medium: contains nutritive supplements for the growth of some organisms, example: BAP
  • Differential medium: distinguishes organisms by differences in their cultural characteristics, example: MacConkey Agar
  • Selective medium: promotes the growth of desirable organisms, example: BSA (Bismuth sulfide agar)
  • Special/Specific culture medium: supports the growth of specific organisms, examples include Ptragnani medium (specific for MTB) and Thayer martin (neisseria group)
• TECHNIQUES OF INOCULATION

  • Liquid culture medium￾Inoculate organisms by shaking Slant medium

  ￾streaking on the bottom of test tube in a zigzag position

  • Butt medium-stabbing

  • Butt /slant- by Stabbing then streaking in zigzag position

  • Plated medium- streaky

  METHODS OF STREAKING PLATED MEDIUM

   Radial streak

   Overlap streak

   Multiple streak

   Interrupted streak

   Multiple interrupted - obtained pure isolated colonies

   Colonies - group of bacteria forming
• TYPES OF COLONIES  Smooth colonies - uniform in texture, easily emulsified in NSS (normal saline solution). Associated with violet organism  Mucoid - exhibit slidy or watery appearance  Rough – granulated and appearance. Difficult to emulsified in NSS
• NUTRITIONAL REQUIREMENTS  Autotrophic/Autotrophs - also called lithotrophs - Requires inorganic substance for growth and survival of bacteria  Heterotrophic/Heterotrops - Also called organictrophs - Requires organic substances for life
• OXYGEN REQUIREMENTS

  Aerobs- organisms heed oxygen

  for growth and survival

  Anaerobs - organisms that live in

  the absence of oxygen
• Strict/obligate aerobes are organisms that can only live in the presence of oxygen, for example, Bacillus Subtilis
• Facultative anaerobes are primarily aerobes but can still grow even in the absence of oxygen, such as Escherichia coli
• Microaerophiles are organisms that need small amounts of oxygen because they die if there's too much oxygen; for example, Yersinia pestis
•  FACULTATIVE ANAEROBS

  Organisms that are primarily aerobs but they can still grow even in the absence of Oxygen

  • Ex: Escherichia coli
• Classification of Bacteria as to Temperature Requirements

  • Psychrophilic - grows at lower temperature (10-20 °C or 5-10 °C)
  • Mesophilic - grows at moderate temperature (39-44 °C)
  • Thermophilic - grows at highest temperature (50-80 °C)
  • Extreme Thermophilic – most optimum growth (above 80 °C)
• Phases of Bacterial Growth

  1. LAG PHASE/PHYSIOLOGICAL: Period of adjustment of bacteria/organisms

  2. EXPONENTIAL: Organisms become active for growth and multiply at the maximum rate, then rapidly increase in cell number through binary fission

  3. STATIONARY PHASE: Phase of equilibrium where the number of organisms roughly equals the number that have died due to lack of nutrients or unfavorable conditions, accumulating toxic debris

  4. DEATH PHASE/DECLINE PHASE: Complete cessation of multiplication with the death rate increasing rapidly

  5. PHASE OF REJUVENESCENCE
• Microaerophiles


  • Yersinia pestis
• Temperature Requirements

  • Optimum temperature: 30-37 °C- best suited for bacterial growth
  • Minimum temperature wherein organisms live: 10-20°C
  • Maximum temperature where organisms live: 40-45°C
  • Thermal death point - temperature which in a given time, destroy all the organisms present
  • Thermal death time - length of time that needed to kill organisms present in a given substance at a given place
• Microaerophiles


  • Organisms that need small amounts of oxygen because organisms die if there's too much oxygen
  • Needed oxygen: 2-10 °C