Bacteria cultivation

• Cultivation of bacteria


  The process of growing microorganisms in culture by taking bacteria from the infection site (i.e., the in vivo environment) and growing them in the artificial environment of the laboratory (i.e., the in vitro environment)

  
• Purposes of bacterial cultivation

  • To grow and isolate all bacteria present in a clinical specimen
  • To determine which of the bacteria that grow are most likely causing infection and which are likely contaminants or colonizers (normal microbiota)
  • To obtain sufficient growth of clinically relevant bacteria to allow identification, characterization, and susceptibility testing

  
• Culture media


  Nutritional needs classification: Fastidious & Non fastidious
• Phases of growth media


  • Broth (liquid)
  • Agar (solid)
  • Biphasic medium that contains both a liquid and a solid phase
• Broth media


  • Nutrients are dissolved in water from clear to turbid (i.e., cloudy)
  • May contain a pH indicator that changes color in the presence of bacterial metabolites
  • Location of growth within the broth provides an indication of the type of organism present based on oxygen requirements
• Agar media


  • Solidifying agent added to the nutrients and water
  • Agarose, the most common solidifying agent, melts at high temperatures (95°C) but resolidifies after the temperature falls below 50°C
  • Stable solid gel referred to as agar
  • Pure colony: all bacterial cells within a single colony are the same genus and species, having identical genetic and phenotypic characteristics (i.e., are derived from a single clone)

  
• Types of culture media

  • Nutritive media (blood or chocolate agars)
  • Selective media
  • Enriched media
  • Enrichment broth
  • Supplemental broth media
• Nutritive media


  • Support the growth of a wide range of microorganisms and are considered nonselective because, theoretically, the growth of most organisms is supported
  • Can also be differential, in that microorganisms can be distinguished on the basis of certain growth characteristics evident on the medium
  • Blood agar is considered both a nutritive and differential medium because it differentiates organisms based on whether they are alpha (a)-, beta (b)-, or gamma (g)-hemolytic

  
• Selective media


  • Support the growth of one group of organisms but not another by adding antimicrobials, dyes, or alcohol to a particular medium
  • Can also be differential media if, in addition to their inhibitory activity, they differentiate between groups of organisms
• Enriched media


  Contain growth enhancers that are added to nonselective agar to allow fastidious organisms to flourish
• Enrichment broth


  A liquid medium designed to encourage the growth of small numbers of a particular organism while suppressing other flora present
• Supplemental broth media


  Broth media can be used as a supplement to agar plates to detect small numbers of most aerobes, anaerobes, and microaerophiles
• Routine primary plating media

  • Nonselective agar plate
  • Enriched medium for fastidious organisms for normally sterile body fluids or a site in which fastidious organisms are expected
  • Selective and differential medium for enteric gram-negative bacilli for most routine bacterial cultures
  • Selective medium for gram-positive organisms for specimens in which mixed gram-positive and gram-negative bacteria are found
  • Additional selective media or enrichment broths for specific pathogens as needed

  
• Media sterilization


  1. Autoclave sterilization at 121°C for at least 15 minutes

  2. Molten agar is allowed to cool to approximately 50°C before being distributed to individual petri plates

  3. Delicate media components can be sterilized by membrane filtration
• Basic aseptic technique


  1. Work area disinfection

  2. Sterilization of loops and needles by heating to red-hot

  3. Flaming of culture tube mouths

  4. Inoculation of liquid and solid media

  5. Final flaming of loops/needles and inoculated tubes

  6. Inoculation of petri plates

  7. Final disinfection of work area
• Inoculation of bacteria


  Streak plate or pour plate methods are used to obtain pure cultures from mixed populations

  
• Identification of bacterial isolates

  • Genotypic identification methods (based on genome analysis)
  • Phenotypic identification methods (based on physical and metabolic characteristics)

  
• Genotypic identification


  Highly specific and sensitive, involves detecting the presence of a gene or a particular nucleic acid sequence unique to the organism
• Phenotypic identification


  Based on observable physical or metabolic characteristics of bacteria, including microscopic morphology, colony morphology, environmental requirements, antimicrobial resistance, nutritional requirements, and biochemical reactions

  
• Macroscopic (colony) morphology


  • Evaluation of colony size, shape, odor, color (pigment), surface appearance, and changes in the surrounding agar medium (e.g., hemolysis)
  • Provides preliminary information but should not be relied upon alone for identification