Working safely with Biological materials

1. Working safely with Biological materials Aseptic technique, sterilization and tissue culture techniques

2. Biological Hazards • Bacteria: food industry, protein and enzyme production • Viruses: DNA transfection into cells of bacteria or higher organisms • Molds & fungi: antibiotic production • Yeasts: complex protein production • Cells of higher organism grown in culture: product production and testing • Whole plants: food production, protein production • Whole animals: product testing, antibody production

3. Minimizing the risks of Biohazards • Standard microbiological practices • Containment: separation of biohazard (organism(s)) and the worker. • Biosafety Levels: BSL1: well characterized strains of microorganisms, do not cause disease BSL2: agents that may cause human disease BSL3 and 4: highly infectious agents

4. Biological Safety Cabinets • Provide containment for aerosols and separate the work material from the operator and the laboratory.

5. Biological Safety Cabinets • HEPA (High Efficiency Particulate Air) filter is a specially constructed filter made of highly pleated glass and paper filters. - remove particles of 0.3um or larger i.e. most bacteria and micro organisms - not effective for removing chemical vapors from air. (Laminar flow cabinet: has a sterile directed air flow, but not all are BSC as some only protect the work bench but not the operator.)

6. Horizontal Hoods • Class I cabinet is designed to protect the operator from airborne material generated at the work surface. It draws air from the room and is vented back to the room after filtering. • In a horizontal hood items should be placed no closer than 3 inches from the back of the hood (nothing should touch the filter).

7. Biological Safety Hood (BSC) • Class IIA BSC do not have external ducts and release filtered air directly into the lab. • Class IIB BSC maintain faster air flow and are ducted to external exhaust systems • (Class III cabinets, also called glove boxes,provide total containment for extremely hazardous biological agents)

8. Proper use of Class II Biosafety cabinets. • Class II cabinets are appropriate for microbiology and tissue culture work. • Many BSC’s contain a UV germicidal lamp i.e. capable of killing bacteria or other microorganisms.

9. Maintaining the sterile field: • Designate a sterile working area in the hood. • Always minimize clutter. Waste and other items should never enter the hood. • Place only sterile items within the sterile field and un-sterile in the “dirty” area. • Never reach across the sterile field. • Open, dispense, and transfer items without contaminating them . • When in doubt about whether something is sterile, consider it contaminated.

10. A direct path must be maintained between the filter and the area inside the hood. • Air downstream from non-sterile objects becomes contaminated from particles blown off these objects, thus critical items should be placed as close to the air source as possible.

11. Sanitization, disinfection and sterilization • Sanitization: achieved by applying antiseptic; general reduction in number of microorganisms • Disinfection: removal of all or almost all pathogenic organisms. • Sterilization: killing of all living organisms Disinfection resistance of organisms • Larger viruses<bacteria<fungi<small viruses<mycobacteria<bacterial spores

12. Sterilizing Solutions • Autoclaving - pressure constant at 15-20 lb/in2 (PSI) - destroys microorganisms and bacteria - test sterility with spore strips or autoclave tape. • Filtration - used for heat sensitive products/solutions - pore size <0.2µm in diameter

13. Storage of Biological Solutions • Bactericidal agents • Decreased temperature - liquid nitrogen - rapid/snap freezing - freeze/thaw cycles - addition of glycerol • Lyophilization • Nonionic detergent

14. Tissue Culture • In vitro propagation of cells from a higher organism • Monolayer of cells covered by a layer of appropriate growth-sustaining liquid medium. • Primary cell cultures – newly isolated cells growing outside the body for the first time. • Cell lines – cells that are established in in long term culture. • Use Class II BSC as sterile work surface and sterile solutions and equipment are essential to avoid contamination with extraneous microorganisms.

15. Uses for cultured cells • Biomedical research: growth control etc. • Sources of DNA for genomic and cDNA libraries • Source of species-specific proteins • Propagation of viruses (e.g. for vaccine production) • Production of recombinant proteins that cannot be produced in bacteria (ie. if extensively post-translationally modified)

16. Immunofluorescent staining