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Filtering andCentrifugation

Physical Separation of

Solids from Liquids

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Part I Filtration

Familiar filtering - funneling Paper filters with

simple funnels

Buchner Funnels Bacteria, fungi,

viruses pass through

easily

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Vacuum filtration

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Replaceable Membranes

Membranes must be

appropriate pore size

Bacteria > 0.3 m Viruses > 0.02 m

(not filterable)

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Depth Filter

Asbestos or glass

fibers.

Tortuous path,particles trapped in

filter

Clarifying solutions

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Membrane filter

Highly polymerizednitrocellulose orpolysulfone

Pore size controlledby polymerizationreaction

Particles (bacteria,

fungi) trapped onsurface, some infilter

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Nucleation track (Nucleopore)

filters

Polycabonate films

Nuclear radiationand chemical etching

cause holes in sheet

Typically sold in 0.2and 0.45 m poressizes

Particles trapped onsurface

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Like this

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Disposable filter units

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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

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Part II Centrifuges, rotors, and

their tubes

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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

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RCF as a function rpm

0

20000

40000

60000

80000

100000

120000

0 5000 10000 15000 20000 25000 30000

rpm

RCF

15 cm

7 cm

3 cm

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Pellets and supernatants from

cultures

Supernatant usually spent

media to be discarded.

Pellet bacterial or yeast cells

to be collected

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cell lysis studies

Supernatant may contain

DNA or other liberated cell

constiituent.

Pellet Cell debris to be

discarded

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DNA precipitation

Supernatant alcohol and salt

used to precipitate DNA

DNA Pellet Warning! DNA

pellets are pretty much

invisible

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Minifuges

14,500 rpm or

14,000 x g

Pellet bacteria Economical, small

foot print

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Microfuges

13,000 rpm or

16,000 x g

More samples,sturdier

Pellet bacteria, can

collect DNA

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Tabletop centrifuges

>20,000 rpm or

>35,000 x g

Widest applications Similar to Avanti

Refrigerated units

preferred to collect

DNA

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Ultracentrifuges

> 100,000 x g

Operate under vacuum air creates heat fromfriction, and slows rotor down

Pellet membranes, ribosomes Used in gradient work

CsCl 24 hour separation of DNA

Sucrose pelleting cell fractions small proteins toribosomes

SvedbergUnits rate of migration through asucrose gradient

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Rotors

Massive storeskinetic energy

Fixed angle Tubes

held at about 45oangle to vertical

Swinging bucket tubes on hinges. At

full speed they goperpendicular togravity

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Conical tubes

Pre-sterilized, plastic

disposable

Maximum force ofonly 6,000 -9,000 x g

Not compatible with

solvents!

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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

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Place your tubes in the rotor

Tubes ofequal mass

opposite oneanother

Hinges up

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Ready to try?

Centri 3

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