Lecture 9 Contamination

 Lecture 9 Contamination

  • Type
    • chemical
      • leaching of chemical from disposable plasticware
      • QA issue
      • inadequate cleaning of glassware
      • cells die
    • biological
      • microbe
      • mycoplasma
      • virus
      • cross-contamination
      • contamination may persist or cell die
  • Consequence
    • financial burden
    • delay progress
    • unreliable data
    • loss of resources
  • Source
    • operator
    • environment
    • equipment
    • reagent
    • cell line
  • Prevention
    • minimize source
    • regular monitor
    • routine test for mycoplasma/virus
    • characterization to confirm cross-contamination
  • Detection & eradication
    • routine screening
    • disposal should be done correctly
    • early detection prevent widespread
  • Mycoplasma
    • class mollicutes
    • small, lack cell wall
    • depend on host to survive
    • grow on host surface/fuse with host
    • do not destroy host
      • common reduce growth rate
      • high titre do not cause morphology change
      • chronic effects→chromosomal abnormalities, increased cloning efficiency & transformation rate
  • PCR
    • detect genotypic changes
    • not all variant but more sensitive
    • target gene→16S rDNA
    • positive control→mycoplasma DNA (510bp)
    • negative control→water, no band, false positive
    • + internal control→2 bands, make sure PCR normal, false negative
  • Direct culture
    • cultured cell in mycoplasma broth 6d
    • plate on nutrient agar 8d
    • positive control→live mycoplasma culture
    • fried egg colony 200um diameter
  • DNA fluorescence
    • less sensitive
    • fixed adherent cells + fluorescent dye DAPI/Hoescht binds to DNA
    • positive control (for staining)→nuclei of cultured cells
    • EN fluorescence
    • false positive due to presence of host RNA or debris
  • Indicator cells
    • adherent cells→enrich mycoplasma growth
    • increase sensitivity
    • prevent false positive due to autofluorescence/debris
    • suspension
    • MDCK, A549
    • adequate cytoplasm
    • spread well in culture
    • free from mycoplasma
  • Other method→>2 method required for detection
  • Eradicate mycoplasma
    • decontamination when valuable cells→experienced operator under quarantine

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