ISOLATION ANS CULTIVATION OF MICROORGANISMS

• Pathogenic


  Capable of causing disease
• Pure culture


  One species of microorganisms
• Mixed culture


  Mixture of 2 or more other species
• Isolation of the organism

  Obtaining a pure culture by separating one species of microbe from a mixture of other species
• Sources for isolating different pathogens

  • Body tissues
  • Fluids; blood, saliva
  • Sputum
  • Pus
  • Feces
  • Spinal fluid
  • Bile
  • Pleural fluids
  • Stomach fluids
• Isolation methods


  1. Streaking

  2. Plating

  3. Dilution

  4. Enriched procedure

  5. Single celled technique
• Streaking


  Most widely used method of isolation; pouring a suitable sterile; allowing the medium to solidify
• Plating


  Diluting of a mixture of microbes until only a few bacteria are left in each milliliter of the suspension
• Dilution


  Method used for microbes which cannot be isolated by streaking or plating method; Predominating form
• Enriched procedure

  Use of media and conditions of cultivation which favor the growth of the desired species, ex: when man suffers from typhoid
• Single celled technique


  Most ideal and difficult method of securing pure culture; Suspension of the pure culture is placed on the under-side of a sterile cover-glass mounted over a moist chamber on the stage of the microscope
• Culture media


  Collectively known as "culture media"—serve as soil in which bacteria are planted for the purpose of study
• Culture media


  • Must contain all the essential nutrients required by the organisms for its growth and reproduction
  • Suitable source of energy, building materials and growth factors must be supplied in adequate amounts
• Types of culture media

  • Defined or Synthetic Media
  • Complex or Non-synthetic Media
  • Living Cells
• Defined or Synthetic Media


  Media prepared from chemical compounds
• Complex or Non-synthetic Media

  Media that are prepared from ingredients that have not been precisely defined
• Living Cells


  Used for cultivation of viruses, ex: fertilized egg incubated for 8-12 days are able to support the growth of many viruses
• 4 Types of Culture Media

  • New Media
  • Derived Media
  • Chemically Defined Media
  • Special Media
• New Media


  Substance occurring in nature (e.g. milk, eggs, blood, extract of plant and animal tissues)
• Derived Media


  Includes known substances by the chemical of each unknown (e.g. nutrient broth, nutrient agar, nutrient gelatin)
• Chemically Defined Media


  Exact chemical composition known
• Special Media


  Includes combination of the other 3 types of media
• 4 Media Used in a Laboratory

  • Enrichment
  • Selective
  • Differential
  • Propagation
• Enrichment Media


  Ingredients prepared to "enhance" the growth of certain microbes, especially the suspected pathogen (e.g. blood agar, chocolate agar)
• Selective Media


  "Inhibit" the growth of unwanted microbes which might be in the specimen
• Differential Media


  Designed to "differentiate" among microbes, where different bacterial species may produce dissimilar colony colours
• Propagation Media


  To "propagate", or keep microbes growing for a long time (e.g. nutrient broth, nutrient agar)
• 3 Main Steps in the Preparation of Media

  • Preparation as solutions of chemicals and adjusting the pH
  • Dispensing the media
  • Sterilization