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