BIOMED NOTES 24/25

 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 ...

 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...

  Cell type tissue of origin morphology specialized fx 1 tissue > 1 cell type specific cell type derived from differ tissue smooth muscle cell from aorta, pulmonary, coronary each cell type variety cell line differ sp finite→primary-senescence continuous→established-tumor Process dissection & tissue isolation tissue disaggregation→detach cells using enzymes, grow in culture vessels enrichment→only cells wanted persist culture→perform characterization to confirm cells subculture max 3 flask finite continuous ethics, identify donors, cooperation from clinicians optimization of culture conditions, detailed records on cell handling=provenance finite→after some subculture, increase til max cell no, drop=senescence continuous=transformed→genotypic & phenotypic changes Research obj ds/condition tissue & cell population or type specific requirement Factors sp finite vs continuous normal vs transformed history growth rate, cell viability, morphology, mode of growth culture re...

 Lecture 7 Serum-free media, Defined media supplements Media development tissue extract, body fluid Eagle MEM→calf, human, horse serum, protein hydrolysate, embryo extract serum-free→facilitate downstream process & reduce risk of adventitious infectious agents Physiochemical pH normal fibroblast 7.4 to 7.7 transformed 7.0 to 7.4 phenol red at 7.4 , orang at 7, yellow at 6.5, lemon yellow <6.5, pink at 7.6, purple at 7.8 buffer open dish →CO2 cause pH high overproduction of CO2 & lactic acid in transformed cell lines at high cell concentrations bicarbonate HCO3 buffer widely use requires CO2 incubation pH 7.2-7.4 inexpensive, non toxic pH fluctuation when expose to air HEPES pH 6.8-8.2 more stable than HCO3 w/o CO2 incubation better pH stability, less sensitive to temp more expensive, potential toxicity at high concentrations phosphate buffer pH 6.8-7.4 less common stable, inexpensive can interfere with certain cellular process maintain optimal pH, critical for cell...

 Lecture 6 Culture Vessels & Substrates Culture Vessels containers or devices used to grow and maintain microorganisms, cells, tissues, or plants in controlled laboratory conditions provide a sterile environment and support the growth of biological samples Types Petri dishes Test tubes Flasks Multiwell plates Roller bottles: Cylindrical bottles that rotate to increase surface area for cell growth. Spinner flasks: Vessels with magnetic stirrers for suspension cultures. Bioreactors: Specialized vessels for large-scale cell or microbial cultures, often with controlled temperature, pH, and oxygen levels. Plant culture vessels: Specialized containers for plant tissue culture, often with ventilation systems. Function Providing sterile environment Supporting cell growth an proliferation Facilitating gas exchange Enabling nutrient supply and waste removal Allowing easy monitoring and manipulation Culture substrate medium or material used to support the growth of microorganisms, cell...

Lecture 5 Safety, Bioethics Validation SOP Safety Biosafety levels 1 well characterized agents not known to cause ds 2 moderate-risk agents moderate danger to personnel & environment 3 indigenous/exotic potential respiratory transmission & serious/lethal infection 4 dangerous exotic agents high risk of life threatening ds, aerosol transmission, unknown risk of transmission, most stringent MSDS General safety trained operator supervisor ensure proper procedure & attire proper disposal of glassware & sharps for incineration Chemical toxicity use liquid detergent, avoid powdered disinfectant hypochlorite bleach skin dimethyl sulphoxide penetrate skin mutagen, carcinogen, cytotoxic drugs in fume hood/class 2 OSH officer had detailed handling method CO2, O2, N2 CO2, N2 cause asphyxiation O2 cause fire evacuation route N2 -196C→frostbite, asphyxiation, explosion use gloves when handling Fire & burns keep alcohol in narrow/plastic bottles, minimize volume do not return bur...