Lecture 4 Aseptic Technique

 Lecture 4 Aseptic Technique

  • Aseptic technique
    • prevent contamination by microbes
    • maintain sterile conditions
    • ensure integrity of results
    • mimic tissue condition of human
    • maintain barrier between cultured cells and potential contaminants
  • Importance
    • reliability
    • contamination can lead to cell death
    • biosafety risk, costly
    • specialized equipment
  • Common contaminants
    • airborne particles
    • microbes
    • non sterile equipment/reagent
    • improper technique
  • How to minimize
    • monitor & detect by visual inspection, microscopy, microbe test
  • Elements
    • quiet→avoid air currents, minimize traffic, dust-free, no sample processing, microbiological or animal work nearby
    • work surface→clean & tidy, bring only required items, swab down surface between procedures, arrange items to avoid crossover
    • personal hygiene→wash hands, tie hair, no talking, wear mask; caps, gowns, masks only in GMP lab
    • reagents & media→swab bottles/container before use
    • cultures→quarantine new cultures, grow separately w/o Ab to confirm no contamination
  • Open bench layout→crescent arrangement, Bunsen burner center
  • Principles
    • sterility maintenance
    • contamination prevention
    • proper handling of sterile materials
  • Sterile handling
    • swabbing
    • capping
    • flaming→disrupt laminar flow, damage HEPA or melt plastic fittings
    • handling bottles and flasks
    • pipetting
    • avoid pouring
  • Types of laminar flow hood
    • horizontal protect cultures; vertical better for operator
    • common class 2
    • class 3, 4→infectious agents
    • routine check & service every 6 months, change filter
    • HEPA monitored by engineers on regular basis
    • regular weekly checks below work surface for spillage & clean up
    • UV for sterilization, use goggles & cover exposed skin
  • Apparatus & equipment
    • regular cleaning
    • no replacement/movement while working
    • disinfect all equipment entering lab
    • humidified incubator=major source of contamination
    • clean with non toxic detergents→Decon
    • swab shelves with alcohol, allow to evaporate

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