1. Filtering and Centrifugation Physical Separation of Solids from Liquids

2. Part I – Filtration Familiar filtering - funneling Paper filters with simple funnels Buchner Funnels Bacteria, fungi, viruses pass through easily

3. Vacuum filtration

4. Replaceable Membranes Membranes must be appropriate pore size Bacteria > 0.3  m Viruses > 0.02  m (not filterable)

5. Depth Filter Asbestos or glass fibers. Tortuous path, particles trapped in filter Clarifying solutions

6. Membrane filter Highly polymerized nitrocellulose or polysulfone Pore size controlled by polymerization reaction Particles (bacteria, fungi) trapped on surface, some in filter

7. 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

8. Like this

9. Disposable filter units

10. 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

11. Part II – Centrifuges, rotors, and their tubes

12. 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

13. RCF as a function rpm 15 cm 7 cm 3 cm

14. Pellets and supernatants from cultures Supernatant – usually spent media to be discarded. Pellet – bacterial or yeast cells to be collected

15. Pellets and supernatants from cell lysis studies Supernatant – may contain DNA or other liberated cell constiituent. Pellet – Cell debris to be discarded

16. Pellets and supernatants from DNA precipitation Supernatant – alcohol and salt used to precipitate DNA DNA Pellet – Warning! DNA pellets are pretty much invisible

17. Minifuges 14,500 rpm or 14,000 x g Pellet bacteria Economical, small foot print

18. Microfuges 13,000 rpm or 16,000 x g More samples, sturdier Pellet bacteria, can collect DNA

19. Tabletop centrifuges >20,000 rpm or >35,000 x g Widest applications Similar to Avanti Refrigerated units preferred to collect DNA

20. 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

21. 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

22. Conical tubes Pre-sterilized, plastic disposable Maximum force of only 6,000 -9,000 x g Not compatible with solvents!

23. 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

24. Place your tubes in the rotor Tubes of equal mass opposite one another Hinges up

25. Ready to try?