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

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

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

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

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.)

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).

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)

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.

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.

 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.

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

Sterilizing Solutions  Autoclaving - pressure constant at lb/in 2 (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

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

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.

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)

Immunofluorescent staining