BACT211 (LAB)

• Aseptic transfer of a culture from one culture vessel to another

  1. No contaminating microorganisms are introduced in the process

  2. Transfer organisms from an isolated colony on a plate of solid medium to a broth tube

  3. Inoculate various media (solid or liquid) from a broth culture for various types of tests
• Aseptic technique


  • Transfer from tube media to tube media
  • Transfer from plate media to plate media
  • Transfer from tube media to plate media (and vice versa)
• Inoculation of bacteria


  • Pure Culture Technique
  • Subculture Technique
  • Evaluation of Slants
• Tube media inoculation


  1. Broth – mix (loop or needle)

  2. Slant – streak (loop or needle)

  3. Deep agar – stab (needle)

  4. Butt-slant – stab then streak (needle)
• Sterilization


  Destruction of all forms of life including bacterial spores
• Disinfection


  Destruction of some forms of life except bacterial spores
• Disinfectant


  Applied to inanimate objects
• Antiseptic


  Applied to skin; cannot be used as disinfectants
• Methods of sterilization


  • Physical methods
  • Chemical methods
• Moist Heat


  Coagulation of bacterial proteins including bacterial enzymes
• Autoclave


  Operates based on the principle of steam under pressure
• Autoclave


  • Effective indication: Sterilization - 121°C for 15lbs/in2 for 15 minutes
  • Effective indication: Decontamination - 135°C for 30lbs/in2 for 30 minutes
  • Biological indicator: Bacillus stearothermophilus
• Tyndallization


  Fractional discontinuous sterilization
• Tyndallization


  • Effective indication: 100°C for 30-60 minutes (commonly used to destroy heat resistant endospores)
  • Instrument: Arnold's Sterilizer
• Inspissation


  Thickening through evaporation
• Inspissation


  • Effective indication: 75-80°C for 2 hours
  • Instrument: Inspissator
• Dry Heat


  Oxidation of bacterial components
• Dry Heat methods


  • Direct Flame
  • Direct/Hot Air Oven
• Dry Heat Oven


  • Effective indication: 160-180°C for 1.5 to 2 hours
  • Biological indicator: Bacillus subtilis var. niger
• Incineration


  Burns materials into ashes; used in the disposable of biological wastes
• Incineration


  • Effective indication: 870-980°C for 2 seconds
• Ionizing Radiation


  Works by alkylation of nucleic acid of bacteria using high energy short wavelength deep penetrating gamma rays; used for heat sensitive materials
• Ionizing Radiation


  • Biological indicator: Bacillus pumilis
• Filtration


  Based on membrane gradient by differences in particle size; used for the sterilization of heat sensitive materials
• Filtration methods


  • Water/Liquid Solutions/Antibiotics/Vaccines
  • Air: High Efficiency Particulate Air Filter (HEPA)
• Water/Liquid Solutions/Antibiotics/Vaccines filtration


  • 0.45-0.80μm – most bacteria, yeasts, and molds are retained but may allow passage of Pseudomonas- like organisms
  • 0.22 μm – used to filter Pseudomonas- like organisms; used for critical sterilization of parenteral solutions
  • 0.01 μm – able to retain small viruses
• Air filtration: High Efficiency Particulate Air Filter (HEPA)


  • Has a pore size of 0.3 μm; usually used in Biological Safety Cabinet (BSC) and rooms of immunocompromised patients
• Chemical methods of sterilization

  • Peracetic Acid
  • Formaldehyde Vapor/Vapor Phase H2O2
  • Glutaraldehyde
  • Ethylene Oxide (ETO) gas
• Ethylene Oxide (ETO) gas

  • Biological indicator: Bacillus subtilis var. globigii
• Boiling


  Destroys vegetative cells of bacteria but not their spores
• Pasteurization methods


  • Batch: 62.5°C for 30 minutes
  • Flash: 72°C for 15 seconds
  • Ultra-High Temperature (UHT): 72°C – 110°C – 72°C for 5 seconds
• Non-ionizing Radiation


  Uses low energy long wavelength ultraviolet rays to disinfect heat sensitive materials as well as large spaces
• Alcohol


  Mechanism of action: dehydration, lipid dissolution, and protein denaturation
• Alcohol


  • 70% Alcohol not 90%
  • Minimum Contact Time: 1-2 minutes or until completely evaporated
• Halogens


  Mechanism of action: inhibits protein function and acts as strong oxidizing agents
• Halogens


  • Chloride (Cl) in NaOCl
  • Iodine (I2) in Betadine
• Heavy metals


  • Mercury (Hg)
  • Copper (Cu)
  • Silver (Ag)
• Heavy metals


  Mechanism of action: denaturation of enzymes and other essential bacterial proteins
• Heavy metals


  • Mercury (Hg): active ingredient or merthiolate
  • Copper (Cu): CuSO4 crystals used as algaecide
  • Silver (Ag): 1% AgNO3 used as prophylactic agent
• Quaternary Ammonium Compounds (QUATS)

  • Zephiran: Benzalkonium chloride
  • Cepacol: Cetylpyridium chloride