Filtering and Centrifugation Physical Separation of Solids from Liquids
Part I – Filtration Familiar filtering - funneling Paper filters with simple funnels Buchner Funnels Bacteria, fungi, viruses pass through easily
Vacuum filtration
Replaceable Membranes Membranes must be appropriate pore size Bacteria > 0.3 m Viruses > 0.02 m (not filterable)
Depth Filter Asbestos or glass fibers. Tortuous path, particles trapped in filter Clarifying solutions
Membrane filter Highly polymerized nitrocellulose or polysulfone Pore size controlled by polymerization reaction Particles (bacteria, fungi) trapped on surface, some in filter
Nucleation track (Nucleopore) filters Polycabonate films Nuclear radiation and chemical etching cause holes in sheet Typically sold in 0.2 and 0.45 m pores sizes Particles trapped on surface
Like this
Disposable filter units
Syringe filters Disposable membrane or Nucleopore filters Filter-sterilizing small volumes of liquids Media, solutions, tissue culture In line filters attach to tubing (pumps) Also can be used for gasses
Part II – Centrifuges, rotors, and their tubes
Centrifugal force Force pressing the particle down relative to the force of gravity (RCF; units are g) Angular velocity expressed in rpm Radius, distance from center of rotation
RCF as a function rpm 15 cm 7 cm 3 cm
Pellets and supernatants from cultures Supernatant – usually spent media to be discarded. Pellet – bacterial or yeast cells to be collected
Pellets and supernatants from cell lysis studies Supernatant – may contain DNA or other liberated cell constiituent. Pellet – Cell debris to be discarded
Pellets and supernatants from DNA precipitation Supernatant – alcohol and salt used to precipitate DNA DNA Pellet – Warning! DNA pellets are pretty much invisible
Minifuges 14,500 rpm or 14,000 x g Pellet bacteria Economical, small foot print
Microfuges 13,000 rpm or 16,000 x g More samples, sturdier Pellet bacteria, can collect DNA
Tabletop centrifuges >20,000 rpm or >35,000 x g Widest applications Similar to Avanti Refrigerated units preferred to collect DNA
Ultracentrifuges > 100,000 x g Operate under vacuum – air creates heat from friction, and slows rotor down Pellet membranes, ribosomes Used in gradient work CsCl – 24 hour separation of DNA Sucrose – pelleting cell fractions small proteins to ribosomes Svedberg Units – rate of migration through a sucrose gradient
Rotors Massive – stores kinetic energy Fixed angle – Tubes held at about 45 o angle to vertical Swinging bucket – tubes on hinges. At full speed they go perpendicular to gravity
Conical tubes Pre-sterilized, plastic disposable Maximum force of only 6,000 -9,000 x g Not compatible with solvents!
Microcentrifuge Tubes Plastic, sterile, disposable centrifuge tubes 2, 1.5, 0.5, and 0.2 (microamp) formats Most molecular techniques, small reaction volumes Special racks and storage
Place your tubes in the rotor Tubes of equal mass opposite one another Hinges up
Ready to try?