Lecture 8 Preparation & Sterilization

 Lecture 8 Preparation & Sterilization

• Sterilization
  • cleaning
    • All new apparatus, materials and instruments should be soaked in detergent overnight, thoroughly rinse and dried (do not expose materials that will corrode to detergent for > 30 min)
    • Used items should be rinsed in tap water and immersed in detergent immediately after use, soak overnight, rinse in deionized water, dry in oven
  • packaging→steam penetration, impermeable to dust, microbe, mite
  • sterilization
    • metal→dry heat
    • silicone rubber PTFE PCO3 cellulose acetate/nitrate filter→autoclave
• Methods
  • 
  • dry heat→160C 1-2h
  • moist heat→121C 20m
  • glass
    • moist heat with loose cap & autoclave tape, autoclave at 121C for 20m
    • slack cap allow steam enter bottle and tighten it after cool down
    • dry heat preferable
    • thermalog indicator turns blue with high temp & steam→safe
    • surface also need to carry correct charge for cell propagation
    • caustic alkaline detergents render surface not suit for cell attachment→neutralize with 0.1M HCl/H2SO4
    • detergent remove residue
    • no toxic residue left behind to leach out into medium/reagent
    • detergent→7X, MP Biomedicals, Decon
    • 
• Alternative
  • Immerse in 70% alcohol for 30 min, dry them off under UV light in a laminar flow hood
  • Ethylene oxide but 2-3 weeks are required to clear it completely from the plastic surface
  • γ- irradiation at 25kGy
• 
• Liquid sterilization
  • avoid accidental inclusion of toxic substance
  • enable reagent to be totally defined & fx of its constituents to be fully understood
  • reduce risk of microbial contamination
  • 
• Reagents & media
  • water purification
    • 1 rinse glassware & reagent prepare→glass distillation, reverse osmosis
    • 2 remove both organic & inorganic colloids→carbon filtration
    • 3 remove ionized inorganic materials→high grade mixed bed deionization
    • final removes microbe, trap resin may escaped from deionizer→micropore filtration
    • ultrapure water if water is recycled continuously from micropore filter to reservoir
• Media preparation
  • commercial
    • working strength solution 1x w or w/o glu→direct use
    • 10x concentrates w/o NaCO3 & glu→dilute first
    • powder w/w/o NaCO3 & glu→cheapest, not sterile, 10x concentrates cost 2-3x powder media but save on sterilization, 1x media most expensive & convenient
  • adjust pH→alkali neutralize 10x concentrated medium
  • +NaCO3→stable equilibrium with atmospheric CO2
  • addition to medium→glu is unstable
  • QC→check by incubate aliquot of complete medium at 37C for 48h before use
• Media sterilization
  • autoclave
    • less labor intensive, less expensive, low failure
    • buffer pH 4.25 with succinate to stabilize VB
    • glu replaced by glutamate/glutamyl dipeptide/added sterile after autoclave
  • sterile filtration
    • for heat labile solution
    • filter thro 0.1-0.2 um PES, PCO3, PTFE, cellulose acetate
    • -+pressure from a pressurized container/peristaltic pump
    • in-line + peristaltic pump 100L
    • bottle-top + vacuum pump 10L
    • large scale 100-10kL