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In vitro techniques.

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Presentation on theme: "In vitro techniques."— Presentation transcript:

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

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