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In vitro techniques
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
Why do you want to use an in vitro technique ?
count bacteria
microbial metabolism and growth
simulate rumen conditionspredict feed quality
protein, fibermicrobial ecologysimulate rumen digestion
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
Types of in vitro systems
batch culture
fed batch culture
semi-continuous culture
continuous culture
In vitro system components
flasksimple to excruciatingly complex
mediumbuffer, substrate, other nutrients
gas phase
flask
Glass is best
Hard plastic
Not red rubber, silicone tubing
buffers
Variations on a themeWeller & Pilgrim, Burroughs, Goering &
Van Soest, Menke, McDougall etc.
Bicarbonate, phosphatepH 6.7 to 6.8 ??
Reducing agents
Anaerobiosis
redox potential, analogous to pHEh in rumen = -300 to 350 mV10-56 molecules O2/LCopper column
O2 soluble in waterBoiling, bubbling with O2 free gas
Oxidized redox cmpds are toxicResazurin at 0.00001%
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
Microbial growth
Growth & death of microbes
Section Phase Growth rate
A Lag Zero
B Acceleration Increasing
C Exponential Constant
D Retardation Decreasing
E Maximum stationary Zero
F Decline Negative
Microbial growth
lag phasevariable with inoculum size, growth phase,
media
log phasehighly reproducible, no substrate limitation
stationary phaseunbalanced growth, no DNA or net RNA
synthesis, smaller cells
Batch culture
pure culture studies
prediction of feed digestibilityTilley & terryGoering & van SoestMenke, gas production
Tilley & Terry (1966)
McDougall’s buffer
2 stage process48 h rumen liquor, 48 h pepsin
DM digestion
Goering & Van Soest (1970)
Modified Tilley & TerryMore complete mediumReducing agent
2 step “true digestibility”
Gas production
Abou Akkada, Menke,Pell, European groups, IwaasaGas production is proportional to
fermentationDependent on pHVent or no-vent ?
In Vitro Gas System – Pressure Transducer
Fed batch
not commonly used
keep organism at or near logarithmic growth for extended periods
particularly good for slow growing organisms, co-cultures
Continuous culture
maintain bacteria at exponential growth for extended periods
growth rate proportional to limiting nutrient addition rate flow rategrowth rate proportional to dilution rate
until critical dilution rate
Semi-continuous culture
more rumen-like than continuous
solid substrates
kinetics more complicated
substitute for cannulated cows
Nakimura & Kurihara
system for protozoa
dialysis membrane
2.3 l volume
90 g/d
Nakimura & Kurihara
Slyter et al.
system for ruminal digestion
simple
500 ml volume
Up to 2.5 volumes/d
40 g/d
Slyter et al.
Rusitec
feed in two bags
1000 ml volume
0.8 to 1.5 volumes/d
24 g dm/d
Rusitec
Hoover et al.
differential flow rates
500 ml volume
up to 3.2 volumes/d
80 to 160 g/d
Hoover et al.
Teather & Sauer
700 ml volume
1.6 volumes/d
30 g DM/d
Designed to maintain protozoa, study rumen ecology
Continuous culture kinetics
Logarithmic growth