In vitro techniques
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Transcript In vitro techniques
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 conditions
predict feed quality
protein, fiber
microbial ecology
simulate 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
flask
simple to excruciatingly complex
medium
buffer, substrate, other nutrients
gas phase
flask
Glass is best
Hard plastic
Not red rubber, silicone tubing
buffers
Variations on a theme
Weller & Pilgrim, Burroughs, Goering &
Van Soest, Menke, McDougall etc.
Bicarbonate, phosphate
pH 6.7 to 6.8 ??
Reducing agents
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%
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 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
Batch culture
pure culture studies
prediction of feed digestibility
Tilley & terry
Goering & van Soest
Menke, gas production
Tilley & Terry (1966)
McDougall’s buffer
2 stage process
48 h rumen liquor, 48 h pepsin
DM digestion
Goering & Van Soest
(1970)
Modified Tilley & Terry
More complete medium
Reducing agent
2 step
“true digestibility”
Gas production
Abou Akkada, Menke,Pell, European
groups, Iwaasa
Gas production is proportional to
fermentation
Dependent on pH
Vent 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 rate
growth 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