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 Bicarbonate, phosphate Reducing agents
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 O2 soluble in water
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 %

12. Reducing agents Resazurin (blue) resorfol (pink)
resorfol (pink) resorfol (clear), 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
Section
Phase
Growth rate A
Lag
Zero B
Acceleration
Increasing C
Exponential
Constant D
Retardation
Decreasing E
Maximum stationary F
Decline
Negative

15. Microbial growth lag phase log phase stationary 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 DM digestion
48 h rumen liquor, 48 h pepsin
DM digestion

18. Goering & Van Soest (1970) Modified Tilley & Terry 2 step
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