1. New Trends in shaking: Improved culture results with new vessels
Dr. Daniel Brücher, Product Manager Infors AG, Switzerland

2. Topics:
Summary previous webinar
Introduction to Thomson flasks
TPP centrifuge tubes
Process optimization

3. Nobel price: antibiotic Streptomycin (USA)
1861
1900
1949
1967
2005
Better growth using a shaker
Bacterial fermentation Static
Hawrylenko patent magnetic drive

5. Oxygen supply is a critical factor!
Oxygen transfer gasphase – liquid phase
Oxygen concentration
in air:
21% = 300 mg/L (norm.) O2
CO2
Gas-phase
 on the LIQUID SURFACE!!!
Funghi
Bacteria
Liquid-phase
Yeast
Mammalian cells
Max. oxygen solubility
in water:
Max. 7 mg/L (37°)
nutrients
waste
Oxygen supply is a critical factor!

6. Function of shaking Suspend cells Mixing of the liquid
Microbes and cells LOVE shaking!!!
Function of shaking
Suspend cells
Mixing of the liquid
Resolve gradients
Increase surface: Enhance gas exchange! O2
CO2

7. x2 Influence factors oxygen transfer: Surface to volume ratio
Large surface per volume: better gas transfer x2 O2
CO2 O2
CO2
Erlenmeyer flask
Fernbach flask

8. Filling volume Very important factor with Erlenmeyer flasks:
Max. 30% (250 ml total  83 ml)
Optimum 25% (250 ml total  62 ml), sometimes 10%
Price to pay  less capacity

9. Shape of the flask Very important factor with Erlenmeyer flasks:
Angled sides reduce liquid movement.

10. „How can I get a better culture result in Erlenemeyer flasks?
Use 10% - 30% max filling volume in Erlenmeyer
new designs  up to 50%
Increase shaking speed as much as possible
Limit „cell“: shear stress
Limit „bact“: flask fixing, spilling
Use 50 mm shaking throw for large flasks, especially cell
Consider other culture vessels!

11. Topics:
Summary previous webinar
Introduction to Thomson flasks
TPP centrifuge tubes
Process optimization

12. Bacterial applications: Ultra Yield FlasksTM and AirOTopTM Seal, Thomson Instrument Company, disposable/re-usable
2.5 L total volume
1.0 L filling volume

13. Efficient sterile filter  Good gas exchange What´s the difference?
Large-diameter neck
Steep side walls
 Large surface/vol ratio
Baffles
 Good mixing
Culture protocols/application notes

14. Efficient sterile filter/Large-diameter neck

15. Why do baffles make a difference?
 They provide good mixing  good oxygen transfer
Recommended shaking speed 2.5 L:
250 rpm (25 mm, bacterial applications)

16. 7.8 L 8 x 2,5 L Ultra Yield (capacity: 12-13) Erlenmeyer:
13 x 2000 per deck
30% filling:
26 L
10% filling:
7.8 L
7 x 5 L Optimum Growth

17. 6 x 2,5 L Ultra Yield flasks
Clamps:
ATM 2000

19. Bacterial applications : Ultra Yield FlasksTM and AirOTopTM Seal, Thomson Instrument Company, disposable Tested by: Genentec, GSK, Pfizer

20. Bacterial applications : Ultra Yield FlasksTM and AirOTopTM Seal, Thomson Instrument Company, disposable Tested by: Genentec, GSK, Pfizer

21. Cell-applications: Optimum Growth FlasksTM, Thomson Instrument Company, disposable

22. Cell-applications: Optimum Growth FlasksTM, Thomson Instrument Company, disposable

23. Cell-applications

25. Cell-applications

26. Field report London: New Brunswick I26. Thomson Ultra Yield. Flasks 2
Field report London: New Brunswick I Thomson Ultra Yield Flasks 2.5 litre mm rpm Bact. application
„However the I26R are a complete nightmare….I am trying to return the NBS shakers, as the company cannot get the shaker to work with the Ultra -Yield flasks“

27. Field report London: Multitron. Ultra Yield Flasks, 2
Field report London: Multitron Ultra Yield Flasks, 2.5 litre 3deck Multitron 25 mm Number of flasks: 12 Filling volume: 1 litre Shaking speed: 250rpm (up to 300 OK as well)

28. Recommended shaking speed: 250 rpm (25 mm) Maximum: 300 rpm (25 mm)
6 x 2,5 L Ultra Yield flasks
Clamps:
ATM 2000
Recommended shaking speed:
250 rpm (25 mm)
Maximum:
300 rpm (25 mm)

29. Topics:
Summary previous webinar
Introduction to Thomson flasks
TPP centrifuge tubes
Process optimization

30. Application: centrifuge tubes „TubeSpin® bioreactor“

31. Application: centrifuge tubes „TubeSpin® bioreactor“

32. Application: centrifuge tubes „TubeSpin® bioreactor“

33. Application: centrifuge tubes „TubeSpin® bioreactor“

34. Application: centrifuge tubes „TubeSpin® bioreactor“
Always 50 mm throw!
150 – 250 rpm (50mm)!

35. Shaking speed
20mL
50mm
150rpm
20mL
50mm
240rpm

36. Topics:
Summary previous webinar
Introduction to Thomson flasks
TPP centrifuge tubes
Process optimization

37. Bacterial applications : Ultra Yield FlasksTM and AirOTopTM Seal, Thomson Instrument Company, disposable Tested by: Genentec, GSK, Pfizer

38. Increase limiting factor  better growth
Optimize: always look at the WHOLE system!
Limiting factor!
Increase limiting factor  better growth

39. Limiting factor! Optimize: always look at the WHOLE system!
Further increase of oxygen:
little effect

40. Optimize: always look at the WHOLE system!
LB medium („Luria broth“):
5 g/L yeast extract
10 g/L Trypton
0.5 – 10 g/L NaCl
TB medium („Terrific broth“):
24 g/L yeast extract
12 g/L Trypton
4 mL Glycerin
Phosphate buffer

41. Optimize shaking cultures: Look at the WHOLE system!
Shake flask = black box
Limiting factor = ???
Optimization: usually try and error
Increasing oxygen alone may not be enough
Enriched culture medium may be required

42. Summary: Thomson flasks: - double volume AND - better culture results
Multitron Pro can handle the load!
Cheaper shakers do not reach the shaking speed
Argument for Infors shakers.
Process optimization:
Always look at the whole system
Use enriched medium

43. Thank you!