Filtering and Centrifugation

Physical Separation of Solids from Liquids

Part I – FiltrationFamiliar 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

))()(1012.1( 25 rxF 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

0

20000

40000

60000

80000

100000

120000

0 5000 10000 15000 20000 25000 30000

rpm

RC

F

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 45o 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 rotorTubes of equal mass opposite one another

Hinges up

Ready to try?