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Taùc giaû thaønh thaät caûm ôn nhöõng yù kieán ñoùng goùp veà nhöõng thieáu soùt.
University of Tropical
Agriculture
Thu Duc, Ho Chi Minh City
Vietnam
Laboratory
Manual
J. :Ly and Nguyen Van Lai
UTA Ecolaboratory
1997
Preface
These
instruction are for the use in the LABORATORY
and should be seen as Manual for every - day work. The equipments and technique
use in this manual very simple and easy to use. Supply for any one who want to
research.
Table of contents
Dry
matter
HCN
Amoniac
Nitrogen
NDF
ASH
Disolved oxygen
In vitro ileal digestibility
In vitro gas production
pH
Wash value
Water soluble solids
Preparation of the
sample
The
sample preparation serves several purpose
1/
To get the sample ready for grinding
2/
to get it in the proper physical state standardized particel size
3/
To get the sample suitable for storage
It
is a big advantage to pre - dry all samples because the grinding will be much
easier and the samples will not deteriorate during storage.
The
first step of the sample preparation is the mixing of the whole sample brought
to the laboratory. This includes in some cases chopping. Take care not to get
any separation of the fine particels during this mixing step.
Determination of dry matter (DM)
The
principle of the method is that, by heating a certain sample, it is possible to
eliminate all its water content, and then dry material is obtained. This
technique necessitates a balance and a heating device, usually an oven.
Instructions
1/
Weight a dry and clean container.
2/
Add the original, fresh sample to the container, and weight both of them
together. This
operation is made at least in duplicates of the same sample.
3/
Dry the sample to constant weight, which implies that all the water has been
eliminated.
4/
Weight the dry sample in the container.
Calculations
% DM = (DS/OS) x 100
Taking into account the weight of the container (C), then
OS = Weight of the original sample, and (OS + C)- C = OS
DS = Weight of the dry sample, and (DS + C) - C = DS
Example
The
weigh of a sample of fresh leaves plus a container was 15.12g and 13.40g in the
fresh (original) and dry state, whereas the weight of the container was 12.88 g.
Then :
OS = 15.12 - 12.88 = 2.24 g
DS = 13.40 - 12.88 = 0.52 g
Therefore,
% DM = (0.52/ 2,24) x 100 - 23.21 %
The
DM content from a duplicate of the same sample was 22.8%. Then the average DM
content of the sample of leaves was.
Average
% DM = (23.21 + 22.87)/2 = 23.04.
Principle :
Procedure :
Weigh
of the samples ( fresh leaf about 20 grams, dry leaf about 5 grams)
and then put in to flask kieldalh add 250 - 300 ml distill water and add
8 ml Chloroform after that boiling them.
Put
8 ml potassium hydroxyt 0.1 N into a 250 ml E - flask and place it on the upper
shelf with receive tube dipped in the potassium. When there is 100 ml in the E -
flask it OK.
Titration
with Nitrate silver 0.1 N the colour from white in to chase white colour
Calculation
:
V * 0.005204
% HCN = ----------------------
* 100
Weight
V : ml silver nitrate
titration
0.005204 : 1 ml silver nitrate equivalent
0.005204 g HCN
Determination of Amoniac
Principle :
Procedure :
-
Weight of the samples and then put in to the kieldalh
add about 5 g Oxyt magie
add 300 ml distill water after that boiling them.
-
Using 250 ml triangle bottle add 50
ml boric acid 4% collect amoniac until 200 - 250 ml
and then titration with sulfuric acid 0.1 N. The colour from white in to
initial pink
Calculation :
V * 0.0017
% Amoniac = ----------------
* 100
Weight
V : ml acid titration
0.0017 : constant 1 ml acid equivalent 0.0017 g
amoniac
Determination of Nitrogen
Prepare
chemical
-
Salt mixture : K2SO4 : CuSO4. 5H2O in to 10 : 1 proportion
Take 1000g K2SO4 and mix well with 100g CuSO4.5H2O
-
Sulphuric acid for digestion : Concentrated, ind. Grade is enough
-
Sodium hydroxide : 40% NaOH in tap water = 10 N
Dissolve 2kg NaOH in tap water and make up to 5 litter
-
Mix indication : Methylenblue and Methyl red
Dissolve 0.12g Methylen blue in 100 ml Ethanol
Dissolve 0.270g Methyl red in 200 ml Ethanol
Mix the two!
*Boric
acid + indicator
2% boric acid in water + mix indicator
Dissolve 100 g of boric acid in water and make up to 5 litter.
Add 25 ml of mixed indicator.
*
Sulphuric acid for titration 0.1 N
Prepare a 1,00 N (0.5 M) H2SO4 from aspulle if possible.
Dilute it 10 times. Take 100 ml and make up to 1000 ml
Principles
Procedure
Digestion
Weigh
accurately samples and put samples in to kieldald
flask add 10 - 15 g of this salt mixture and add 25 ml acid sulphuric
concentration and then put the flask on the heaters
and boiling them.
Digest
until the colour has turned green it Ok
Allow
the flask to cool down for at least 30 min
Add
250 ml of distill water
Distillation
Put
50 ml of boric acid + indicator solution in to a 250 - 300 ml E - flask and
place it on the upper shelf with the receiver tube dipped in the acid
Pour
careful 90 - 100 ml of NaOH 40% in the flask
connect
the flask to the distiller and then shake it carefully. This has to be done as
quickly as possible to avoid lossed of nitrogen
When
there is 150 ml in the E-flask, lift it down to the lower shelf, continue the
distillation until there is 175 ml in the E-flask
Wait
for 5 - 10 minute so the last distillate can come down into the E-flask
Titration
and calculation
Titration
with 0.1 N H2SO4 until the colour is between green and purle
correct
the titer with the blank - value
Two
blanks should be run every time a new preparation of boric acid is used and also
when you start using a new bulk of any of the chemicals.
Calculation
(Titer - blank x 0.875)
% CP = ----------------------------
Sample weight (g)
(Titer - blank x 0.14)
% Nitrogen = --------------------------
Sample
weight (g)
0.1 x 0.014 x 6.25 x 100
* 0.875 = -------------------------------
1000
* 0.1 = The
strength of the acid for titration
* 14 =
Formula weight for nitrogen
* 6.25 = Factor to transfer
from nitrogen to protein on most feed samples
* 1000 = Conversion from liters to milli - liters
* 100 = To get %
Note :
Always use protectives like lab, coats, gloves and spectacles when working with
strong acids and alkalines.
Determination of NDF
Prepare
chemicals for NDF
Bottle
1 : 150 g sodium dodecyl sulfate + 50 ml Tri - ethylenglycol + 2 litter water
Bottle
2 : 93.05g EDTA + 34.05g sodium tetraborate + 1 litter water
Bottle
3 : 22.8g Disodium hydrogen phosphate + 1 litter water gently boiling and cool
down
Bottle
4 = put 2 and 3 into 1 adiust until 5 litters and correct pH 6.9 - 7.0 it OK
Principle
Procedure
Weigh
accurate 1 - 1.5g of the grinded sample into a beaker (600ml) or flask. Add 100
ml of NDF - solution and 2 ml of decalin.
Heat
to boil and reflux for 60 minute
Place
a previously tared glassfillter - crucible on the filtermaniflold. Transfer
contents of beaker to the crucible while using low vacuum from the beginning.
Rinse beaker with hot water and also the residue in the crucible with hit water.
Rinse finally twice with acetone. Dry crucible at 105oC over night. Weigh out
after cooling down in a desiccator. Ash at 500oC for 2 - 3 hours. Cool down in
desiccator and weigh again.
OS - DR
% WL (DM) = -------------- x
100
OS
Weigh
after drying - Tare weight
NDF (INCL ASH) % = ------------------------------------------ x 100
Sample weight
Weigh
after drying - weigh after ASH
NDF (EXCL ASH) % =
-------------------------------------------------- x 100
Sample weight
Determination of total
ASH
Weigh
accurately ( 1 mg) 1 - 5 grams of sample into a tare porcelain crucible. Ash the
material until greyish ash results in an electric furnace at 550 oC - 600 oC .
The time it takes depends on the sample and the furnace, normally 3 - 4 hours.
Cool the crucible and its contents in a desiccator and weigh. If the ash is not
greyish you could treat it with hot water, filter through, ashfree filterpaper,
wash with hot water and transfer the filterpaper to a crucible and ash again for
5 - 10 min, then cool in a desiccator and weigh
Calculation
Weight of sample after ashing
ASH % = -------------------------------------------
x 100
Weight
of sample before ashing
Determination of dissolved oxygen
The
oxygen which is dissolved in water is determined by a portable disolved oxygen
meter (DO meter) from Hanna instruments (HI 8543). The principle of the
determination is based on polarography. The DO meter has a disolved oxygen probe
consisting of a membrane covering the polarographic sensor and a built-in
thermistor for temparature measurements and compensation. Dissolved oxygen can
be estimated in the range of 0.0 to 19.9 mg/l with a resolution of 0.1mg/l and
an accuracy of 0.2 mg/l.
Instructions
1/
Connect the DOmeter with the DO probe and switch on. Wait about 10 minutes to
ensure
the probe has been polarized.
2/
Prepare the electrolyte solution by adding 15 to 20 drops of HI 7041 solution
into 20 ml of
distilled water.
3/
Wet the sensor by soaking the bottom of the probe in the dilute electrolyte
solution for 5
minutes.
This
is done by unscrewing the protective red and black plastic cap. Ensure the
o-ring is properly placed inside the membrane. Rinse the membrane cap with
electrolyte while shaking it gently. Refill with fresh electrolyte. Gently tap
the sides of the membrane with the finger tip to ensure that no air bubbles
remain. To avoid damage, do no tap the membrane directly on the bottom. With the
sensor facing down, firmly screw the membrane cap clock -wise to the end of the
threads. Some electrolyte will overflow.
4/
For slope calibration, rinse the probe throughly (particularly after the zero
calibration) with
some water and blot dry in a manner as not to damage the membrane (use
cloth or soft
paper). Place the probe in vertical position in air. Wait a few minutes
until the
measurement stabilizes.
Press and hold the red button between the two calibration screws. The
decimal point should disappear
from the display. Using a small screwdriver, turn the slope trimmer until the
display reads 100. Release the red button and the display will show the
saturation value at
that ambient temperature. Saturation value is the maximum dissolvable
oxygen value in a
liquid at that temperature.
5/
Dissolved oxygen is determined in the samples after slope calibration. All the
measurements are referred to
sea level and zero salinity..
In vitro ileal digestibility of dry matter (DM), N and
organic matter (OM) in pigs.
Principle
The
in vitro estimation of ileal digestibility permits the evaluation of the quality
of feeds, specially N and amino acids for pigs. It has been well established the
straight correlation which does exits between in vitro ileal and in vitro
(pepsin/ pancreatin), and in turn, the interdependence between in vitro ileal N
digestibility and performance traits of economic importance in pigs.
The
methods herein described is that proposed by Dierick et al (1985) fully
developed in tropical non conventional feeds at the swine Research Institute at
Havana by the research team of Ly (see for example, Dominguez et al 1996).
Procedure
1/
Weight an amount of dry and ground sample containing 150 mg of protein (N x
6.25) and
put it into a 100 ml erlenmeyer flask. Add 20 ml of a pepsin solution (
1mg enzyme/`ml
HCL 0.075 N) plus a drop of timerosal.
2/
Incubate the sample in a water bath at 37oC (preferably 80 strockes per minute)
during 4
hours.
3/
Neutralize until pH = 7.5 with NaOH (or KOH) 0.2N ( about 7.5 ml).
4/
Add 20 ml of a solution of 15% pancreatin in a phosphate buffer. The phosphate
buffer
can be prepare by mixing 10 of a solution of 31.50g KH2PO4.3H2O/ L and 90
ml of
another solution of 45.6g K2HPO4/ L. The solution is adjusted to pH = 7.5
and made up to
a L with distilled water. Repeat the incubation in a water bath at 37oC
as in 2.
5/
Add a solution of sodium tungstate (5%) to precipitate the undigested protein.
6/
Transfer the solution quantitatively to a 120 ml centrifuging tube and
centrifugue the
sample during 10 minutes at 1250 rpm. As alternative, filter
quantitatively, in a previously
weighed filter paper.
7/
Dry to constant weight to determine the dry insoluble residue
8/
Determine the ash content in other dry insoluble residue to calculate the
insoluble organic
matter.
9/
Determine the N content by the Kjeldhal procedure in another dry insoluble
residue.
Calculation
OS - DR
% IVDDM = ---------------
x 100
OS
Where :
* OS = Weight of the original sample, in dry basis
* DR = Weight of the dry residue
When
the in vitro digestibility of organic matter (IVDOM) or in vitro digestibility
of N (IVDM) is to be estimated, calculate by the same procedure for example, the
IVDN should be calculated by the following equation.
NOS -N DR
% IVDN = -------------------
x 100
N
OS
Where :
* NOS = mg of N in the original sample
* NDR = mg of in the dry residue.
References
Dierick,
N, Vervaeke, I., Decuypere, J. and Henderickx, H. 1985. Protein digestion in pig
measured in vitro and in vitro. In : Proc, 3rd Int. Seminar Dig.
Physiol. Pig (A. Just, H. Jogensen and J. A. Fernandez, ed.) Kobenhavn p 329 -
332.
Dominguez,
P. L., Molinet, Y. and Ly, J. 1997. Ileal and in vitro digestibility in the pig
of three floating aquatic macrophytes. Livest. Res. Rural Devel. 8(4) : 37 - 44.
Determination of the in vitro gas production.
The
in vitro gas production (IVDP) technique measures the evolution of gases
(methane and carbon dioxyde) which are produced as end products of fermentation
in the rumen. Production of carbon dioxyde is partly from the fermentation and
partly as result of formation of short chain fatty acids which expels carbon
dioxyde from the bicarbonate buffer solution.
The
method is important for feed evaluation, particularly to predict animal
performance such as feed digestibility and intake in the ruminant. It has the
advantage of the in vitro technique a great number of samples can be evaluate at
the same time, it is time saving and cheaper than those methods requiring the
use of animals, thus becoming expensives by feed cost.
The
IVGP technique provides a great advantage in that the fermentation take place in
a glass syringe which allow for several measurements to be made in the same by
measuring the gas volume at different intervals of time. This means that not
only the possible extent of fermentation can be measured.
The
procedure herein described is that of Menke and Steingass (1988)
Instroduction
1/
All substrates should e milled using a 1mm screen. Weigh 200 mg substrate into
each
(numbered) syringe and record actual weight. Include a blank (for
example. Rumen fluid/
buffer mixture on its own) at the beginning, in the middle of the set,
and at the end. A
sample of hay can be milled and used as a control by including a syringe
with the hay at
the beginning and the end of each rum. Samples should be done in
duplicate or triplicate.
After the weighings are completed, grease the plungers with vaseline, and
place in
incubator at 38oC. This is normally done the day before the run.
2/
Measure distilled water, buffer solution, macromineral solution, micromineral
solution
and resazurin solution into a round, flat bottomed, flask - warm to 38oC.
Then add
reducing solution of sodium sulphide. Place it in a small water bath on
magnetic stirrer,
put magnet into flask and gently bubble carbon dioxyde through the
solution until the blue
color turn to pink, then clear. This means the buffer solution is now
reduced. Raise the
carbon dioxyde tube so that it will be above the level of the buffer/
rumen fluid mixture,
but providing a stream of carbon dioxyde into the flask throughout the
dispensing
procedure. The pH of buffer should be 7 - 7.3.
3/
Collect rumen fluid from the donnor animal, strain through gauze into a warm
beaker, the
final ratio of rumen buffer fluid should be 1:2 Mix rumen fluid in beaker
and transfer to
flask with buffer solution. Make sure the magnet is mixing properly
during the whole
process of dispensing the buffer/ rumen fluid mixture into the syringes.
Add 30 ml to each
syring using the dispenser (do 2 - 3 times 30 ml amounts into a beaker at
the beginning to
be sure the dispenser is properly charger). Fill the syringe, then open
the clip and gently
push the plunger of the syringe so that all the gas is removed. Record
the level in each
syringe and place it in the water bath.
Times
of reading can be chosen to suit the type of substrate in the syringes. For
forages 3 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours and 96 hours
are suitable but for concentrate type substrate it may be necessary to take more
reading in the first 24 hours. It is advisable to gently mix each syringe
2 - 3 the lectures made at 24 hours and 48 hours.
Gas
production is estimated in ml/ 0.2 g of sample per 48 hours.
Preparation of
solution
1/
Macromineral solution.
Na2HPO4
5.7 g
KH2PO4
6.2 g
MgSO4. 7H2O 0.6
g
Make up to one liter with distilled water
2/
Buffer solution
NaHCO3 35 g
(NH4)HCO3
4 g
Make up to one liter with distilled water
3/
Micromineral solution
CaCL2. 2H2O
13.2 g
MnCL2. 4H2O
10.0 g
CoCL2.
6H2O
1.0 g
FeCL2. 6H2O
0.8 g
Make up to 100 ml with distilled water
4/
Reducing solution
NaOH 1N
2 ml
Distilled water
47.5 ml
Na2.7H2O
0.285 g
5/
Resazurin solution
Resazurin 0.100 g
Make up to 100 ml with distilled water
6/
Preparation of the buffer solution
Distilled water
474 ml
Macromineral solution (number 1)
237 ml
Buffer solution (number
2)
237 ml
Micromineral (number 3
)
0.12 ml
Resazurin solution (number 5) 1.22
ml
Warm to 38oC, then add reducing solution (number 4), prepared fresh for
each run.
Determination of pH
The
pH value in different types of samples is determined by a portable digital
pHmeter. The principle of the determination is based on potentiometry. Thee
pHmeter has a pH combination electrode and pH values can be estimated in the
range of 0.00 to 14.00 with a resolution of 0.01 and accuracy of 0.01.
Instructions
1/
Connect the pHmeter with the pH electrode and switch. Set the mode switch to thr
pH
position. This will activate the liquid cristal display. Condition the
electrode in buffer 7.00
until the display is fully stable (about 30 minutes). Give a stirring
action to the electrode
each time it is placed in a new solution.
2/
Adjust the calibration control to make the display read the value of buffer
7.00. Rinse the
electrode with distilled water and blot dry.
3/
Inmerse the electrode in buffer 4.00. When the display is stable (30 seconds),
adjust the
slope control to make the display read the value of buffer 4.00. The pH
value are 6.99 and
4.01 for buffer 7.00 and 4.00 respectively at 30oC. Error may be minimum
in the case that
pH value are not adjust to the temperature of the solution to be tested.
The apparatus is now calibration to read samples with pH 0 - 14.
4/
Rinse the electrode with distill water, blot dry and inmerse in the saple. The
sample must
be homogenous. When the display is stable (30 seconds), it will read the
pH of the sample
Repeat this step for continued samples. Periodically check calibration,
at least one per day.
5/
When all samples are completed, keep the electrode inmerse in distilled water.
The
electrode shall never be kept dry, otherwise electrode damage shall be
irreversible.
Determination of the wash value
Washing
losses or a wash value of a sample are considered to be the amount of water
soluble substances readily utilizable by the animal. This technique was first
applied to ruminant studies concerning the digestibility of roughages.
It now has been suggested to be directly related to the cell contents
(100 - NDF) of a sample, which in turn, could be of application in non ruminant
nutrition studies too.
The
method herein described it that proposed by Ly and Preston (1997) which studies
some detail of the analytical technique, not taken into consideration
previously. This technique requires a balance, an oven and a comercial
semiautomatic washing machine. On the other hand, it is necessary to use nylon
bags of 50 x 50 mm. This bag are made of nylon filter cloth with a pore size of
45 to 55 micrometers, and are available from some catalogue list.
Instructions
1/
Weight a dry and clean bag
2/
Add to the bag, very carefully, 1 g f air - dried sample or 5 g of fresh sample.
Weight again
this operation is made at least in triplicate of the same sample. The
mouth of the bag must
be tightly close, such as to avoid losses during washing. This procedure
can be afford by a
rubber band.
3/
Wash the bag in a comercial, semiautomatic washing macbine of 15 min - cycle,
during 90
min, discarding the water every two cycle. Use tap water at room
temperature. The ratio of
water per bag is of 3 litters.
4/
After washing, let the bag to drain and dry it in an oven to constant weight
5/
Determine the wash value taking into account the original dry matter of the
sample.
6/
The sample is followed for determining the wash value of nitrogen in the sample.
In this
opportunity, the N content in the original and in the washed sample
should be used for
calculations.
Calculations
OS - DR
% WL (DM) = -------------- x
100
OS
Where
OS is the weight in g of the original sample, in dry basis, and DR is the weight
in g of the dry residue.
If
it is desire to determine the wash value of N in the sample, then the
calculation will be as follows
NS - NR
% WL (N) = -------------- x 100
NS
Where
NS and NR in the nitrogen content determined in the original sample and in the
residue obtain after washing. The nitrogen content is usually expressed in
percent in dry basis, in both cases.
Determination of water soluble solids
Water
soluble solids (WSS) are estimated by refractometry, taking into account that a
straight interdependence exists between brix and refractive indices in certains
solutions. A hand refractometer (Fisher Scientific, catalogue No. 13 - 946 -
60A) will be used for WSS determination in leaves and sugar cane juice. The
measuring range of the instrument is 0 - 32% (accuracy, 0.2%)
Calibration
procedure
1/
Keep distilled water and instrument at room temperature (20oC) for 1 hours.
2/
Open the prism cover and put one or two drops of distilled water on the prism.
Close the
prism gently.
3/
Direct the prism towards a light source and look at the scale through the
eyepiece.
4/
Rotate the view control to a clean image of the scale. Real the scale. It the
scale is correct,
the boundary line meets with “0" position. No calibration is
needed. IF the boundary line is
out of “0" position, calibration the boundary line to indicate
“0" position by turning the
calibration screw with a screwdriver.
5/
Calibration is completed wipe off the distilled water from the prism with a soft
clean cloth
or tissue paper.
Instructions.
1/
Open the prism cover and put one or two drops of the sample in the prism.
2/
Close the prism cover gently
3/
The sample volume must cover the prism surface entirely
4/
Direct the prism towards a light source and look at the scale through the
eyepiece
5/
Rotate the view control to focus a clean image of the scale. Real the scale
where the
boundary line interrupts it.
6/
After every measurement, wipe the sample from the prism with a soft cloth of
tissue paper
and water. Keep the prism clean and dry to get the correct reading.
7/
Calculate WSS content (Brix %) from the determined refractive indice (nD), see
table 1
Table 1
: Brix (%) vs refractive index (nD)
Brix
(%) Refractive indices (nD)
Brix (%) Refractive
indices (nD)
0
1.33299
17
1.35890
1
1.33423
18
1.36053
2
1.33588
19
1.36218
3
1.33733
20
1.36384
4
1.33880
21
1.36551
5
1.34027
22
1.36719
6
1.34176
23
1.36888
7
1.34027
24
1.37059
8
1.34477
25
1.3723
9
1.34629
26
1.3740
10
1.34783
27
1.3758
11
1.34937
28
1.3775
12
1.35093
29
1.3793
13
1.35250
30
1.3811
14
1.35408
31
1.3829
15
1.35567
32
1.3847
The
hand refractometer is designed to be used at 20oC, and when used above 20oC, the
reading value (brix %) have to be correcting referring to table 2 by adding the
following value.
Table 2
: Temperature compensation table
Reading
(Brix %)
Temperature (oC)
0
5
10
5
20
25
30
35
25
0.33 0.35
0.36 0.37
0.38 0.38
0.39 0.39
26
0.40 0.42
0.43 0.44
0.45 0.46
0.47 0.47
27
0.48 0.50
0.52 0.53
0.54 0.55
0.55 0.55
28
0.56 0.57
0.60 0.61
0.62 0.63
0.63 0.63
29
0.64 0.66
0.68 0.69
0.72 0.72
0.72 0.72
30
0.72 0.74
0.77 0.78
0.79 0.80
0.80 0.81