Filtering and Centrifugation

Physical Separation of Solids from Liquids

Familiar filtering - funneling

Part I Filtration

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

F (1.12 x10 )( )( r )
5 2
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

120000 100000 80000

15 cm

RCF

60000 40000 20000 0

7 cm
3 cm

5000

10000

15000 rpm

20000

25000

30000

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 rotor

Hinges up

Tubes of equal mass opposite one another

Ready to try?