In vitro techniques

1. In vitro techniques

2. In vivo Techniques • In vivo= in life • Fistula = a hole • Cannula = a device • Ruminant cannula in: esophagus,rumen, abomasum, duodenum, ileum, cecum • Non ruminant cannula in: duodenum, ileum, cecum

3. Why do you want to use an in vitro technique ? • count bacteria • microbial metabolism and growth • simulate rumen conditions • predict feed quality • protein, fiber • microbial ecology • simulate rumen digestion

4. Rumen in vitro techniques • The use of an artificial system to mimic a natural dynamic microbial ecosystem • Always a trade-off between simplicity and precision of mimicry

5. Types of in vitro systems • batch culture • fed batch culture • semi-continuous culture • continuous culture

6. In vitro system components • flask • simple to excruciatingly complex • medium • buffer, substrate, other nutrients • gas phase

7. flask • Glass is best • Hard plastic • Not red rubber, silicone tubing

8. buffers • Variations on a theme • Weller & Pilgrim, Burroughs, Goering & Van Soest, Menke, McDougall etc. • Bicarbonate, phosphate • pH 6.7 to 6.8 ?? • Reducing agents

9. Anaerobiosis • redox potential, analogous to pH • Eh in rumen = -300 to 350 mV • 10-56 molecules O2/L • Copper column • O2 soluble in water • Boiling, bubbling with O2 free gas • Oxidized redox cmpds are toxic • Resazurin at 0.00001%

12. Reducing agents • Resazurin (blue) resorfol (pink) • resorfol (pink) resorfol (clear), -.042 mV • cysteine-HCl cystine, -340 mV • dithiothreitol, -330 mV • sulfide s, -571 mV • titanium citrate, -430 mV • ascorbic acid, -320 mV

13. Microbial growth

14. Growth & death of microbes

15. Microbial growth • lag phase • variable with inoculum size, growth phase, media • log phase • highly reproducible, no substrate limitation • stationary phase • unbalanced growth, no DNA or net RNA synthesis, smaller cells

16. Batch culture • pure culture studies • prediction of feed digestibility • Tilley & terry • Goering & van Soest • Menke, gas production

17. Tilley & Terry (1966) • McDougall’s buffer • 2 stage process • 48 h rumen liquor, 48 h pepsin • DM digestion

18. Goering & Van Soest (1970) • Modified Tilley & Terry • More complete medium • Reducing agent • 2 step • “true digestibility”

23. Gas production • Abou Akkada, Menke,Pell, European groups, Iwaasa • Gas production is proportional to fermentation • Dependent on pH • Vent or no-vent ?

26. In Vitro Gas System – Pressure Transducer

31. Fed batch • not commonly used • keep organism at or near logarithmic growth for extended periods • particularly good for slow growing organisms, co-cultures

32. Continuous culture • maintain bacteria at exponential growth for extended periods • growth rate proportional to limiting nutrient addition rate • flow rate • growth rate proportional to dilution rate until critical dilution rate

33. Semi-continuous culture • more rumen-like than continuous • solid substrates • kinetics more complicated • substitute for cannulated cows

34. Nakimura & Kurihara • system for protozoa • dialysis membrane • 2.3 l volume • 90 g/d

35. Nakimura & Kurihara

36. Slyter et al. • system for ruminal digestion • simple • 500 ml volume • Up to 2.5 volumes/d • 40 g/d

37. Slyter et al.

38. Rusitec • feed in two bags • 1000 ml volume • 0.8 to 1.5 volumes/d • 24 g dm/d

39. Rusitec

40. Hoover et al. • differential flow rates • 500 ml volume • up to 3.2 volumes/d • 80 to 160 g/d

41. Hoover et al.

43. Teather & Sauer • 700 ml volume • 1.6 volumes/d • 30 g DM/d • Designed to maintain protozoa, study rumen ecology

45. Continuous culture kinetics

46. Logarithmic growth