1972L0199 — EN — 22.07.1993 — 003.001 — 1
This document is meant purely as a documentation tool and the institutions do not assume any liability for its contents
►B THIRDCOMMISSIONDIRECTIVE
of27April1972
establishingCommunitymethodsofanalysisfortheofficial controloffeedingstuffs
(72/199/EEC)
(OJ L 123, 29.5.1972, p. 6)
Amended by:
Official Journal
No page date
►M1Commission Directive 81/680/EEC of 30 July 1981 L 246 32 29.8.1981
►M2Commission Directive 84/4/EEC of 20 December 1983 L 15 28 18.1.1984
►M3Commission Directive 93/28/EEC of 4 June 1993 L 179 8 22.7.1993
Corrected by:
►C1Corrigendum, OJ L 320, 27.11.1980, p. 43 (72/199/EEC)
▼B
THIRDCOMMISSIONDIRECTIVE
of27April1972
establishingCommunitymethodsofanalysisfortheofficialcontrol
offeedingstuffs
(72/199/EEC)
THE COMMISSION OF THE EUROPEAN COMMUNITIES,
Having regard to the Treaty establishing the European Economic
Community;
Having regard to the Council Directive of 20 July 1970 (1) on the intro-
duction of Community methods of sampling and analysis for the
official control of feedingstuffs, and in particular Article 2 thereof;
Whereas that Directive requires that official controls of feedingstuffs
be carried out using Community methods of sampling and analysis for
the purpose of checking compliance with requirements arising underthe provisions laid down by law, regulation or administrative actionconcerning the quality and composition of feedingstuffs;
Whereas Commission Directives No 71/250/EEC (
2) of 15 June 1971
and No 71/393/EEC (3) of 18 November 1971 have already established
a number of Community methods of analysis; whereas, in view of
progress made in subsequent work, a third set of methods should be
adopted;
Whereas the measures provided for in this Directive are in accordance
with the Opinion of the Standing Committee for Feedingstuffs,
HAS ADOPTED THIS DIRECTIVE:
Article 1
The Member States shall require that analyses for official controls of
feedingstuffs as regards their levels of starch, crude protein, crude
protein which can be dissolved by pepsin and hydrochloric acid, offree and total gossypol and as regards pepsin activity be carried outusing the methods described in Annex I to this Directive.
Article 2
The Member States shall require that analyses for official controls of
feedingstuffs, for the purpose of detecting and identifying antibioticsof the tetracycline groupand also for determining the levels of chlorte-
tracycline, oxytetracycline, tetracycline, oleandomycin, tylosin, and
virginiamycin in feedingstuffs, be carried out using the methodsdescribed in Annex II to this Directive.
Article 3
The Member States shall, not later than 1 July 1973, bring into force
the laws, regulations or administrative provisions necessary to complywith this Directive. They shall forthwith inform the Commissionthereof.1972L0199 — EN — 22.07.1993 — 003.001 — 2
(1) OJ No L 170, 3.8.1970, p. 2.
(2) OJ No L 155, 12.7.1971, p. 13.
(3) OJ No L 279, 20.12.1971, p. 7.▼M1
▼B
▼M1
▼B
▼B
Article 4
This Directive is addressed to the Member States.1972L0199 — EN — 22.07.1993 — 003.001 — 3
▼B
ANNEX I
1.DETERMINATIONOFSTARCH
Polarimetric method
1.Purposeandscope
This method makes it possible to determine the levels of starch and of high
molecular weight starch degradation products in feedingstuffs, with the
exception of those feedingstuffs which contain beet chips, beet pulp, dried
beet tops or leaves, potato pulp, dehydrated yeast, products rice in inulin
(e.g. chips and meal of Jerusalem artichokes) or greaves.
2.Principle
The method comprises two determinations. In the first, the sample is treated
when hot with dilute hydrochloric acid. After clarification and filtration the
optical rotation of the solution is measured by polarimetry.
In the second, the sample is extracted with 40 % ethanol. After acidifying the
filtrate with hydrochloric acid, clarifying and filtering, the optical rotation is
measured as in the first determination.
The difference between the two measurements, multiplied by a known factor,
gives the starch content of the sample.
3.Reagents
3.1 25 % (w/w) hydrochloric acid, d: 1·126.3.2 1·128 % (w/v) hydrochloric acid.
The concentration must be checked by titration using a sodium hydro-
xide solution 0·1 N in the presence of 0·1 % (w/v) methyl red in 94 %
(v/v) ethanol. 10 ml = 30·94 ml of NaOH 0·1 N.
3.3 Carrez solution I: dissolve 21·9 g of zinc acetate Zn (CH
3COO)2·2H2O
and 3 g of glacial acetic acid in water. Make upto 100 ml with water.
3.4 Carrez solution II: dissolve 10·6 g of potassium ferrocyanide K4[Fe
(CN)6]·3H2O in water. Make upto 100 ml with water.
3.5 40 % (v/v) ethanol, d: 0·948 at 20ºC.
4.Apparatus
4.1 250 ml Erlenmeyer flask with standard ground-glass joint and with reflux
condenser.
4.2 Polarimeter or saccharimeter.
5.Procedure
5.1Preparation of the sample
Crush the sample until it is fine enough for all of it to pass through a
0·5 mm round-meshed sieve.
5.2Determination of the total optical rotation (P or S)(see observation 7.1)
Weigh 2.5 g of the crushed sample to the nearest mg and place in a
100 ml graduated flask. Add 25 ml of hydrochloric acid (3.2), shake to
obtain even distribution of the test sample and add a further 25 ml of
hydrochloric acid (3.2). Immerse the flask in a boiling water bath
shaking vigorously and steadily for the first three minutes to prevent
the formation of agglomerates. The quantity of water in the water bath
must be sufficient for the bath to remain at boiling point when the flask
is introduced into it. The flask must not be taken out of the bath whilst
being shaken. After exactly 15 minutes, remove from the bath, add 30 ml
of cold water and cool immediately to 20ºC.
Add 5 ml of Carrez solution I(3.3)and shake for one minute. Then add
5 ml of Carrez solution II (3.4) and shake again for one minute. Make
upto volume with water, homogenize and filter. If the filtrate is not
perfectly clear (which is rare), repeat the determination using a larger
quantity of Carrez solutions I and II, for example 10 ml.1972L0199 — EN — 22.07.1993 — 003.001 — 4
▼B
Measure the optical rotation of the solution in a 200 mm tube with the
polarimeter or saccharimeter.
5.3Determination of the optical rotation (P ′or S ′)of substances soluble in
40 % ethanol
Weigh 5 g of the sample to the nearest mg, place in a 100 ml graduated
flask and add about 80 ml of ethanol (3.5) (see observation 7.2). Leave
the flask to stand for 1 hour at room temperature; during this time, shake
vigorously on six occasions so that the test sample is thoroughly mixed
with the ethanol. Make upto volume with ethanol (3.5), homogenize and
filter.
Pipette 50 ml of the filtrate (= 2·5 g of the sample) into a 250 ml Erlen-
meyer flask, add 2·1 ml of hydrochloric acid (3.1) and shake vigorously.
Fit a reflux condenser to the Erlenmeyer flaskand immerse the latter in a
boiling water bath. After exactly 15 minutes, remove the Erlenmeyer
flask from the bath, transfer the contents to a 100 ml graduated flask,
rinsing with a little cold water, and cool to 20 ºC.
Clarify using Carrez solutions I (3.3) and II (3.4), make upto volume
with water, homogenize, filter and measure the optical rotation as indi-
cated in the 2nd and 3rd paragraphs of 5.2.
6.Calculationofresults
The starch content as a percentage of the sample is calculated as follows:
6.1Measurement by polarimetry
Percentage of starch ¼2000 P /C0P0ðÞ
α½/C13820º
D
where:
P = total optical rotation in degrees;
P′ = optical rotation in degrees of substances soluble in
40 % ethanol;
α½/C13820º
D = specific optical rotation of the pure starch. The
conventionally accepted values for this factor are
the following:
+ 185·9º: rice starch,
+ 185·4º: potato starch,+ 184·6º: maize starch,
+ 182·7º: wheat starch,
+ 181·5º: barley starch,
+ 181·3º: oat starch,
+ 184·0º: other types of starch and starch mixtures
in compound feedingstuffs.
6.2Measurement by saccharimetry
Percentage of starch ¼
2000
α½/C13820º
D/C12N/C10/C1665 ðÞ S/C0S0ðÞ =¼26/C16N S /C0S0ðÞ
α½/C13820º
D
where:
S = total optical rotation in saccharimetric degrees;
S′= optical rotation in saccharimetric degrees of substances soluble
in 40 % ethanol;
N = weight in g of sucrose in 100 ml of water giving an optical
rotation of 100 sacchararimetric degrees in a 200 mm tube.
The weight varies as follows according to the type of sacchari-
meter used:
16·29 g for French saccharimeters,
26·00 g for German saccharimeters,
20·00 g for other saccharimeters;
α½/C13820º
D= specific optical rotation of the pure starch (see 6.1).1972L0199 — EN — 22.07.1993 — 003.001 — 5
▼C1
▼B
▼B
6.3Repeatability
The difference between the results of two parallel determinations carried
out on the same sample must not exceed 0·4, in absolute value, for starch
contents of less than 40 %, and 1 %, in relative value, for starch contents
of 40 % or more.
7.Observations
7.1 If the sample contains more than 6 % of carbonates, calculated in terms
of calcium carbonate, they must be destroyed by treatment with ab
exactly appropriate quantity of dilute sulphuric acid before determination
of the total optical rotation.
7.2 In the case of products with a high lactose content, such as powdered
milk serum or skimmed milk powder, proceed as follows after adding
80 ml of ethanol (3.5). Fit a reflux condenser to the flask and immerse
the latter in a water bath at 50 ºC for 30 minutes. Leave to cool and
continue the analysis as indicated in 5.3.
2.DETERMINATIONOFCRUDEPROTEIN
1.Purposeandscope
This method makes it possible to determine the crude protein content of
feedingstuffs on the basis of the nitrogen content, determined according to
the Kjeldahl method.
2.Principle
The sample is digested by sulfuric acid in the presence of a catalyst. The acid
solution is made alkaline with sodium hydroxide solution. The ammonia is
distilled and collected in a measured quantity of sulfuric acid, the excess of
which is titrated with a standard solution of sodium hydroxide.
3.Reagents
3.1 Potassium sulfate.
3.2 Catalyst: copper (II) oxide CuO or copper (II) sulfate pentahydrate,
CuSO4·5 H2O
3.3 Granulated zinc.
3.4 Sulfuric acid, ρ20= 1,84 g/ml.
3.5 Sulfuric acid c(½H2SO4) = 0,5 mol/l.
3.6 Sulfuric acid c(½H2SO4) = 0,1 mol/l.
3.7 Methyl red indicator; dissolve 300 mg of methyl red in 100 ml of
ethanol, σ= 95-96 % (v/v)
3.8 Sodium hydroxide solution (Technical grade may be used) β=4 0g /
100 ml (m/v: 40 %).
3.9 Sodium hydroxide solution c = 0,25 ml/l.
3.10 Sodium hydroxide solution c = 0,1 mol/l.
3.11 Granulated pumice stone, washed in hydrochloric acid and ignited.
3.12 Acetanilide (m.p. = 114 ºC, N = 10,36 %)
3.13 Sucrose (nitrogen free).
4.Apparatus
Apparatus suitable for performing digestion, distillation and titration
according to the Kjeldahl procedure.
5.Procedure
5.1Digestion
Weigh 1 g of the sample to the nearest 0,001 g and transfer the sample
to the flask of the digestion apparatus. Add 15 g of potassium sulfate
(3.1.), an appropriate quantity of catalyst (3.2) (0,3 to 0,4 g of copper
(II) oxide or 0,9 to 1,2 g of copper (II) sulfate pentahydrate), 25 ml of
sulfuric acid (3.4) and a few granules of pumice stone (3.11) and mix.1972L0199 — EN — 22.07.1993 — 003.001 — 6
▼M3
▼M3
Heat the flask moderately at first, swirling from time to time if necessary
until the mass has carbonized and the foam has disappeared; then heat
more intensively until the liquid is boiling steadily. Heating is adequate
if the boiling acid condenses on the wall of the flask. Prevent the sides
from becoming overheated and organic particles from sticking to them.
When the solution becomes clear and light green continue to boil for
another two hours, then leave to cool.
5.2Distillation
Add carefully enough water to ensure complete dissolution of the
sulfates. Allow to cool and then add a few granules of zinc (3.3).
Place in the collecting flask of the distillation apparatus an exactly
measures quantity of 25 ml of sulfuric acid (3.5) or (3.6) depending on
the presumed nitrogen content. Add a few drops of methyl red indicator
(3.7).
Connect the digestion flask to the condenser of the distillation apparatus
and immerse the end of the condenser in the liquid contained in the
collecting flask to a depth of at least 1 cm (see observation 8.3). Slowly
pour 100 ml of sodium hydroxide solution (3.8) into the digestion flask
without loss of ammonia (see observation 8.1).
Heat the flask until the ammonia has distilled over.
5.3Titration
Titrade the excess sulfuric acid in the collecting flask with sodium
hydroxide solution (3.9) or (3.10) depending on the concentration of the
sulfuric acid used, until the end point is reached.
5.4Blank test
To confirm that the reagents are free from nitrogen, carry out a blank
test (digestion, distillation and titration) using 1 g of sucrose (3.13) in
place of the sample.
6.Calculationofresults
The content of crude protein is calculated according to the following
formula:
V0/C0V1 ðÞ /C2 c/C20;014/C2100/C26;25
m
Where,
Vo= Volume (ml) of NaOH (3.9 or 3.10)
used in the blank test.
V1= Volume (ml) of NaOH (3.9 or 3.10)
used in the sample titration.
c = Concentration (mol/l) of sodium
hydroxide (3.9 or 3.10).
m = Mass (g) of sample.
7.Verification ofthemethod
7.1Repeatability
The difference between the results of two parallel determinations carried
out on the same sample must not exceed:
0,2 % in absolute value, for crude protein contents of less than 20 %;
1,0 % relative to the higher value, for crude protein contents from 20 %
to 40 %;
0,4 % in absolute value, for crude protein contents of more than 40 %.
7.2Accuracy
Carry out the analysis (digestion, distillation and titration) on 1,5 to 2,0 g
of acetanilide (3.12) in the presence of 1 g of sucrose (3.13); 1 g aceta-
nilide consumes 14,80 ml of sulfuric acid (3.5). Recovery must be at
least 99 %.1972L0199 — EN — 22.07.1993 — 003.001 — 7
▼M3
8.Observations
8.1 Apparatus may be of the manual, semi-automatic or automatic type. If
the apparatus requires transference between the digestion and distillation
steps, this transfer must be carried out without loss. If the flask of the
distillation apparatus is not fitted with a dropping funnel, add the sodium
hydroxide immediately before connecting the flask to the condenser,
pouring the liquid slowly down the side.
8.2 If the digest solidifies, recommence the determination using a larger
amount of sulfuric acid (3.4) than that specified above.
8.3 For products with a law nitrogen content, the volume of sulfuric acid
(3.6) to be placed in the collecting flask may be reduced, if necessary,
to 10 or 15 ml and made upto 25 ml with water.
3.DETERMINATION OF CRUDE PROTEIN DISSOLVED BY PEPSIN
ANDHYDROCHLORICACID
1.Purposeandscope
This method makes it possible to determine the fraction of crude protein
dissolved by pepsin and hydrochloric acid under defined conditions. It is
applicable to all feedingstuffs.
2.Principle
The sample is heated for 48 hours at 40 ºC in a solution of pepsin hydro-
chloride. The suspension is filtered and the nitrogen content of the filtrate
determined according to the method for the determination of crude protein.
3.Reagents
3.1 Hydrochloric acid, d: 1·125.
3.2 Hydrochloric acid 0·075 N.3.3 2·0 U/mg pepsin; pepsin activity is defined in the method described in
Part 4 of this Annex and must be established according to that method.
3.4 About 0·2 % (w/v) freshly prepared solution of pepsin in hydrochloric
acid (3.2): activity: 400 U/l.
3.5 Anti-foaming emulsion (eg silicone).3.6 All the reagents listed under 3 in the method for the determination of
crude protein.
4.Apparatus
4.1 Water bath or incubator, set at 40 ºC ± 1 ºC.
4.2 Kjeldahl digestion and distillation apparatus.
5.Procedure
5.1Preparation of solution (see observation 7.2)
Weigh 2 g of the sample to the mearest mg and place in a 500 ml grad-
uated flask. Add 450 ml of pepsin hydrochloride solution (3.4)
previously heated to 40 ºC and shake to prevent the formation of
agglomerates. Check that the pH of the suspension is less than 1·7. Place
the flask in the water bath or incubator (4.1) and leave there for 48
hours. Shake after 8, 24 and 32 hours. After 48 hours, add 15 ml of
hydrochloric acid (3.1), cool to 20 ºC, make upto volume with water
and filter.
5.2Digestion
Take 250 ml of the filtrate and place in the flask of the distillation appa-
ratus (4.2). Add the reagents necessary for digestion indicated in thesecond sentence of 5.1 of the method for the determination of crude
protein. Homogenize and bring to the boil. If any foam should form,
add a few drops of anti-foaming emulsion (3.5). Continue to boil vigor-
ously until the water has been almost completely evaporated. Reduce the
heat and carefully eliminate the last traces of water.1972L0199 — EN — 22.07.1993 — 003.001 — 8
▼B
▼B
When the solution becomes clear and colourless (or light green if a
copper-based catalyst is used), continue to boil for another hour. Leave
to cool.
5.3Distillation and titration
Proceed as indicated in 5.2 and 5.3 of the method for the determination
of crude protein.
5.4Blank test
Carry out a blank test applying the same procedure but omitting the
sample to be analyzed.
6.Calculationofresults
Subtract the volume of sulphuric acid consumed in the blank test from that
consumed by the test sample. 1 ml of sulphuric acid 0·1 N corresponds to
1·4 mg of nitrogen.
Multiply the quantity of nitrogen by the factor 6·25. Express the result as a
percentage of the sample.
Repeatability
The difference between the results of two parallel determinations carried out
on the same sample must not exceed:
— 0·4, in absolute value, for contents of less than 20 %;
— 2·0 %, in relative value, for contents of not less than 20 % and not more
than 40 %;
— 0·8, in absolute value, for contents of more than 40 %.
7.Observations
7.1 The values obtained by this method have no direct connection with
digestibility in vivo.
7.2 Products with an oil or fat content exceeding 10 % must first be defatted
by extraction with petroleum ether (B.P. 40 to 60 ºC).
4.ESTIMATIONOFPEPSINACTIVITY
1.Purposeandscope
This method makes it possible to established the activity of the pepsin used
in the determination of crude protein dissolved by pepsin and hydrochloric
acid.
2.Principle
Haemoglobin is treated with pepsin in a hydrochloric acid medium under
defined conditions. The non-hydrolyzed fraction of the protein is precipitated
in trichloroacetic acid. Sodium hydroxide and Folin-Ciocalteu reagent are
added to the filtrate. The optical density of this solution is measured at
750 nm and the corresponding quantity of tyrosine is read from a calibration
curve.
Definition: The unit of pepsin is defined as being the quantity of that enzyme
which, under the conditions of the method, liberates per minute, a quantity of
hydroxyaryl groups which, when stained with the Folin-Ciocalteu reagent,
has an optical density corresponding to that of one µmole of tyrosine stained
in the same manner.
3.Reagents
3.1 Hydrochloric acid 0·2 N.
3.2 Hydrochloric acid 0·06 N.
3.3 Hydrochloric acid 0·025 N.
3.4 5 % solution (w/v) of trichloroacetic acid.3.5 Sodium hydroxide solution 0·5 N.
3.6 Folin-Ciocalteu reagent. Place 100 g of sodium tungstate (Na
2WO4.
2H2O), 25 g of sodium molybdate (Na2MoO4.2 H2O) and 700 ml of water
in a 2 litre round-bottomed flask fitted with a standard ground-glass
joint. Add 50 ml of phosphoric acid (d: 1·71) and 100 ml of concentrated1972L0199 — EN — 22.07.1993 — 003.001 — 9
▼B
hydrochloric acid (d: 1·19), connect a reflux condenser to the flask, bring
to the boil and keepthe solution gently boiling for 10 hours. Leave to
cool, detach the reflux condenser, add 175 g of lithium sulphate
(Li2SO4.2 H2O), 50 ml of water and 1 ml of bromine. Boil for 15 minutes
to eliminate excess bromine.
Leave to cool, transfer the solution to a 1 litre graduated flask, make up
to volume with water, homogenize and filter. No greenish coloration
must remain. Before use, dilute 1 volume of the reagent with 2 volumes
of water.
3.7 Haemoglobin solution: Weigh a quantity of haemoglobin (approx. 2 g of
protein substratum determined according to Anson) corresponding to
354 mg of nitrogen (1) and place in a 200 mlflask fitted with a standard
ground-glass joint. Add a few ml of hydrochloric acid (3.2), connect the
flask to the vacuum pump and shake until the haemoglobin has comple-
tely dissolved. Release the vacuum and, while shaking, add hydrochloric
acid (3.2) to make upto 100 ml. Prepare immediately before use .
3.8 Standard tyrosine solution: Dissolve 181·2 mg of tyrosine in the hydro-
chloric acid (3.1) and make upto 1 litre with the same acid (stick
solution). Take 20·0 ml and dilute to 100 ml with hydrochloric acid
(3.1). 1 ml of this solution contain 0·2 µmole of tyrosine.
4.Apparatus
4.1 Water bath set at 25 ºC ± 0·1 ºC by ultrathermostat.
4.2 Spectrophotometer.4.3 Chronometer, accuracy: 1 second.4.4 pH-meter.
5.Procedure
5.1Preparation of the solution (see observation 7.1)
Dissolve 150 mg of pepsin in 100 ml of hydrochloric acid (3.2). Pipette
2 ml of the solution into a 50 ml graduated flask and make upto volume
with hydrochloric acid (3.3). The pH, checked with the pH-meter, must
be 1·6 ± 0·1. Immerse the flask in the water bath (4.1).
5.2Hydrolysis
Pipette 5·0 ml of haemoglobin solution (3.7) into a test tube, heat to
25 ºC in the water bath (4.1), add 1·0 ml of the pepsin solution obtained
in 5·1 and mix with a glass rod thickened at one end, with about 10
back-and-forth movements. Leave the test tube in the water bath at
25 ºC for exactly 10 minutes, timed from the addition of the pepsin solu-
tion (duration and temperature must be strictly observed). Then add
10·0 ml of trichloroacetic acid solution (3.4) previously heated to 25 ºC,
homogenize and filter through a dry filter.
5.3Development of coloration and measurement of optical density
Pipette 5·0 ml of the filtrate into a 50 ml Erlenmeyer flask, add 10·0 ml
of sodium hydroxide solution (3.5) and, shaking constantly, 3·0 ml of
dilute Folin-Ciocalteu reagent (3.6). After 5 to 10 minutes, determine
the optical density of the solution with the spectrophotometer at 750 nm
in cells 1 cm thick against water.
5.4Blank test
For each determination, carry out a blank test as follows:Pipette 5·0 ml of haemoglobin solution (3.7) into a test tube, heat to
25 ºC in the water bath (4.1), add 10·0 ml of trichloroacetic acid solution
(3.4) previously heated to 25 ºC, homogenize, then add 1.0 ml of the
pepsin solution obtained in 5.1. Mix with a glass rod and leave the test
tube in the water bath (4.1) at 25 ºC for exactly 10 minutes. Homogenize
and filter through a dry filter. Follow the procedure indicated in 5.3.1972L0199 — EN — 22.07.1993 — 003.001 — 10
(1) Determine the nitrogen content by the semi-micro Kjeldahl method (theoretical content:
17·7 % of nitrogen).
▼B
5.5Calibration curve
Place 1·0, 2·0, 3·0, 4·0 and 5·0 ml aliquots of standard tyrosine solution
(3.8), corresponding to 0·2, 0·4, 0·6, 0·8 and 1·0 µmoles of tyrosine
respectively in 50 ml Erlenmeyer flasks. Complete the series with a
reference solution free from tyrosine. Make upthe volumes to 5·0 ml
with hydrochloric acid (3.1). Add 10·0 ml of sodium hydroxide solution
(3.5) and, shaking constantly, 3·0 ml of dilute Folin-Ciocalteu reagent
(3.6). Measure the optical density as indicated in the last sentence of
5.3. Trace the calibration curve by plotting the optical densities against
the quantities of tyrosine.
6.Calculationofresults
From the calibration curve read the quantity of tyrosine, in µmoles, corre-
sponding to the optical density of the coloured solution, corrected on the
basis of the blank value.
The pepsin activity, in µmoles, of tyrosine at 25 ºC, per mg and per minute,
is calculated by using the formula:
Units per mg U =mgðÞ ¼0/C132 a
P
where:
a = quantity of tyrosine, in µmoles, read from the calibration curve;
p = weight in mg of the quantity of pepsin added in 5.2.
7.Observations
7.1 The quantity of pepsin to be dissolved must be such that, on final photo-
metric measurement, an optical density of 0·35 ± 0·035 is obtained.
7.2 Two units per mg obtained by this method correspond to:
3·64 Anson milliunits /mg (µmoles of tyrosine/mg · min at 35·5 ºC) or
36400 commercial units/g (µmoles of tyrosine/g in 10 min at 35·5 ºC).
5.DETERMINATIONOFFREEANDGOSSYPOL
1.Purposeandscope
This method makes it possible to determine the levels of free gossypol, total
gossypol and chemically related substances in cottonseed, cottonseed meal
and cottonseed cake and in compound feedingstuffs containing these
substances where more than 20 ppm are present.
2.Principle
The gossypol is extracted in the presence of 3-aminopropan-1-ol, either with
a mixture of propan-2-ol and hexane, for the determination of free gossypol,
or with dimethylformamide, for the determination of total gossypol. The
gossypol is converted by aniline into gossypol-dianiline, the optical density
of which is measured at 440 nm.
3.Reagents
3.1 Propan-2-ol-hexane mixture: mix 60 parts by volume of propan-2-ol
A.R. with 40 parts by volume of n-hexane.
3.2 Solvent A: Place in a 1 litre graduated flask approximately 500 ml of
propan-2-ol-hexane mixture (3.1), 2 ml of 3-aminopropan-1-ol, 8 ml of
glacial acetic acid and 50 ml of water. Make upto volume with the
propan-2-ol-hexane mixture (3.1). This reagent is stable for one week.
3.3 Solvent B: Pipette 2 ml of 3-aminopropan-1-ol and 10 ml of glacial
acetic acid into a 100 ml graduated flask. Cool to room temperature
and make upto volume with N, N-dimethylformamide. This reagent is
stable for one week.
3.4 Aniline A.R.: If the optical density in the blank test exceeds 0·022 , distil
the aniline over zinc dust, discarding the first and last 10 % fractions of
the distillate. Refrigerated and stored in a brown, stoppered glass flask,
this reagent will keepfor several months.
3.5 Standard gossypol solution A: Place 27·9 mg of gossypol acetate in a
250 ml graduated flask. Dissolve and make upto volume with solvent
A (3.2). Pipette 50 ml of this solution into a 250 ml graduated flask
and make upto volume with solvent A. The gossyp ol concentration of1972L0199 — EN — 22.07.1993 — 003.001 — 11
▼B
this solution is 0·02 mg per ml. Leave to stand for one hour at room
temperature before use.
3.6 Standard gossypol solution B: Place 27·9 mg of gossypol acetate in a
50 ml graduated flask, Dissolve and make upto volume with solvent B
(3.3). The gossypol concentration of this solution is 0·5 mg per ml.
Standard gossypol solutions A and B will remain stable for 24 hours if
protected from the light.
4.Apparatus
4.1 Mixer (tumbler): approximately 35 rpm.4.2 Spectrophotometer.
5.Procedure
5.1Test sample
The amount of test sample used depends on the presumed gossypol
content of the sample. It is preferable to work with a small test sample
and a relatively large aliquot part of the filtrate, so as to obtain sufficient
gossypol for precise photometric measurement to be possible. For the
determination of free gossypol in cottonseed, cottonseed meal and
cottonseed cake, the test sample should not exceed 1 g; for compound
feedingstuffs, it may be as much as 5 g. A 10 ml aliquot part of filtrate
is suitable in most cases; it should contain 50 to 100 µg of gossypol. For
the determination of total gossypol, the test sample should be between
0·5 and 5 g, that a 2 ml aliquot part of filtrate will contain 40 to 200 µg
of gossypol.
The analysis should be carried out at a room temperature of about 20 ºC.
5.2Determination of free gossypol
Place the test sample in a ground-necked 250 ml flask, the bottom of the
flask having been covered with crushed glass. Using a pipette, add 50 ml
of solvent A (3.2), stopper the flask and mix for one hour in the mixer.
Filter through a dry filter and collect the filtrate in a small ground-
necked flask. During filtration, cover the funnel with a watch glass.
Pipette identical aliquot parts of filtrate containing 50 to 100 µg of
gossypol into each of two 25 ml graduated flasks (A and B). If neces-
saty, make upthe volume to 10 ml with solvent A (3.2). Then make
the contents of flask (A) up to volume with the propan-2-ol-hexane
mixture (3.1). This solution will be used as a reference solution against
which to measure the sample solution.
Pipette 10 ml of solvent A (3.2) into each of two other 25 ml graduated
flasks (C and D). Make the contents of flask (C) upto volume with the
propan-2-ol-hexane mixture (3.1). This solution will be used as a refer-
ence solution against which to measure the blank test solution.
Add 2 ml of aniline (3.4) to each of flasks (D) and (B). Heat for 30
minutes over a boiling water bath to developthe colour. Cool to room
temperature, make up to volume with the propan-2-ol-hexane mixture
(3.1), homogenize and leave to stand for one hour.
Determine the optical density of the blank test solution (D) by compar-
ison with the reference solution (C), and the optical density of the
sample solution (B) by comparison with the reference solution (A), in
the spectrophotometer at 440 nm using 1 cm glass cells.
Substract the optical density of the blank test solution from that of the
sample solution (= corrected optical density). From this value calculate
the free gossypol content as indicated in 6.
5.3Determination of total gossypol
Place a test sample containing 1 to 5 mg of gossypol in a 50 ml gradu-
ated flask and add 10 ml of solvent B (3.3). At the same time, prepare a
blank test, placing 10 ml of solvent B (3.3) in another 50 ml graduated
flask. Heat the two flasks for 30 minutes over a boiling water bath. Cool
to room temperature and make the contents of each flask up to volume
with the propan-2-ol-hexane mixture (3.1). Homogenize and leave to
settle for 10 to 15 minutes, then filter and collect the filtrates in
ground-necked flasks.
Pipette 2 ml of the sample filtrate into each of two 25 ml graduated
flasks, and 2 ml of the blank test filtrate into each of two other 25 ml1972L0199 — EN — 22.07.1993 — 003.001 — 12
▼B
flasks. Make the contents of one flask from each series upto 25 ml with
the propan-2-ol-hexane mixture (3.1). These solutions will be used as
reference solutions.
Add 2 ml of aniline (3.4) to each of the other two flasks. Heat for 30
minutes over a boiling water bath to developthe colour. Cool to room
temperature, make up to 25 ml with the propan-2-ol-hexane mixture
(3.1), homogenize and leave to stand for one hour.
Determine the optical density as indicated in 5.2 for free gossypol. From
this value calculate the total gossypol content as indicated in 6.
6.Calculationofresults
Results may be calculated either from the specific optical density (6.1), or by
reference to a calibration curve (6.2).
6.1From the specific optical density
The specific optical densities, under the conditions described, will be the
following:
free gossypol: E1%
1c m¼625
total gossypol: E1%
1c m¼600
The free or total gossypol content of the sample is calculated by using
the following formula:
% gossypol ¼E/C11250
E1%
1c m/C1p/C1a
where:
E = corrected optical density, determined as indicated in 5.2;
p= test samp le in g;a = aliquot part of the filtrate in ml.
6.2From a calibration curve
6.2.1 Free gossypol
Prepare 2 series of five 25 ml graduated flasks. Pipette aliquots of
2·0, 4·0, 6·0, 8·0 and 10·0 ml of standard gossypol solution A (3.5)
into each series of flasks. Make upthe volumes to 10 ml with
solvent A (3.2). Complete each series with a 25 ml graduated flask
containing only 10 ml of solvent A (3.2) (blank test).
Make the volume of the flasks in the first series (including the
flask for the blank test) up to 25 ml with the propan-2-ol-hexane
mixture (3.1) (reference series).
Add 2 ml of aniline (3.4) to each flask in the second series
(including the flask for the blank test). Heat for 30 minutes over
a boiling water bath to developthe colour. Cool to room temp era-
ture, make up to volume with the propan-2-ol-hexane mixture
(3.1), homogenize and leave to stand for one hour (standard
series).
Determine as indicated in 5.2 the optical density of the solutions in
the standard series by comparison with the corresponding solutions
in the reference series. Trace the calibration curve by plotting the
optical densities against the quantities of gossypol (in µg).
6.2.2 Total gossypol
Prepare six 50 ml graduated flasks. In the first flask place 10 ml of
solvent B (3.3), and in the others 2·0, 4·0, 6·0, 8·0 and 10·0 ml ofstandard gossypol solution B (3.6) respectively. Make the contents
of each flask upto 10 ml with solvent B (3.3). Heat for 30 minutes
over a boiling water bath. Cool to room temperature, make up to
volume with the propan-2-ol-hexane mixture (3.1) and homoge-
nize.
Place 2.0 ml of these solutions in each of two series of six 25 ml
graduated flasks. Make the contents of the flasks in the first series
up to 25 ml with the propan-2-ol-hexane mixture (3.1) (reference
series).1972L0199 — EN — 22.07.1993 — 003.001 — 13
▼B
Add 2 ml of aniline (3.4) to each flask in the second series. Heat
for 30 minutes over a boiling water bath. Cool to room tempera-
ture, make up to volume with the propan-2-ol-hexane mixture
(3.1), homogenize and leave to stand for one hour (standard
series).
Determine as indicated in 5.2 the optical density of the solutions in
the standard series by comparison with the corresponding solutions
in the reference series. Trace the calibration curve by plotting the
optical densities against the quantities of gossypol (in µg).
6.3Repeatability
The difference between the results of two parallel determinations carried
out on the same sample must not exceed:
— 15 %, in relative value, for gossypol contents of less than 500 ppm;
— 75 ppm, in absolute value, for contents of not less than 500 ppm and
not more than 750 ppm;
— 10 %, in relative value, for contents of more than 750 ppm.1972L0199 — EN — 22.07.1993 — 003.001 — 14
▼B
ANNEX II
1.DETECTION AND IDENTIFICATION OF ANTIBIOTICS OF THE
TETRACYCLINEGROUP
1.Purposeandscope
This method makes it possible to detect and identify antibiotics of the tetra-
cycline group in feedingstuffs containing at least 0.1 ppm of antibiotics, in
concentrates and in premixes.
2.Principle
The sample is extracted with a mixture of methanol and hydrochloric acid.
The extract and reference solutions for comparison are subjected to ascending
paper chromatography. The antibiotics are detected and identified by
comparing their Rf values with those of the standard substances, either by
fluorescence in UV light (high antibiotic contents) or by bioautography on
an agar medium inoculated with B. cereus.
3.Reagentsandculturemedium
3.1Buffer solution, pH 3.5
Citric acid monohydrate A.R. 10·256 gdiSodium hydrogen phosphate Na
2HPO4·
2H2O A.R.7·45 g
Acetone A.R. 300 ml
Distilled water to 1000 ml
3.2Phosphate buffer solution, pH 5.5
Potassium dihydrogen phosphate KH2PO4A.R.130·86 g
diSodium hydrogen phosphate Na2HPO4·
2H2O A.R.6·947 g
Distilled water to 1000 ml
3.3 Eluent I: Mixture of pure nitromethane/pure chloroform/1,3-dichloro-
propan-2-ol: 20/10/1·5 by volume. prepare immediately
before use.
3.4 Eluent II: Mixture of pure nitromethane/pure chloroform/2-picoline:
20/10/3 by volume. Prepare immediately before use.
3.5 Mixture of pure methanol/hydrochloric acid (d: 1·19): 98/2 by volume.3.6 Hydrochloric acid 0·1 N.
3.7 Ammonia, d:0·91.
3.8 Standard substances: chlortetracycline, oxytetracycline, tetracycline, the
activity of which is expressed in terms of hydrochloride.
3.9Micro-organism: B. cereus ATCC No 11.778
Maintenance of the parent strain, preparation of the spore suspension
and inoculation of the culture medium: follow the directions given in
3.1 and 3.2 of the method for the determination of chlortetracycline,
oxytetracycline and tetracycline contents by diffusion on agar which is
described in Part 2 of this Annex.
3.10Culture medium (
1)
Glucose 1 g
Tryptic peptone 10 gMeat extract 1·5 g1972L0199 — EN — 22.07.1993 — 003.001 — 15
(1) Any commercial culture medium of similar composition and giving the same results
may be used.
▼B
Yeast extract 3 g
Agar 20 g
Distilled water to 1000 ml
Adjust the pH to 5·8 immediately before use.
3.11 0·1 % (w/v) 2,3,5-triphenyltetrazolium chloride solution and 5 % (w/v)
glucose solution.
4.Apparatus
4.1 Apparatus for ascending paper chromatography (height of paper: 25 cm).
Schleicher and Schüll paper (2040b or 2043b) or equivalent.
4.2 Centrifuge.4.3 Incubator set at 30 ºC.4.4 U.V. lampfor the detection of fluorescence.4.5 Glass plates approximately 20 × 30 cm for bioautography.
5.Standardsolutions
5.1Stock solutions
Using hydrochloric acid (3.6), prepare from the standard substances (3.8)
solutions with concentrations corresponding to 500 µg per ml of chlorte-
tracycline-HCl, of oxytetracycline-HCl, and of tetracycline-HCl.
5.2Reference solutions for detections by UV light
Dilute the solutions (5.1) with the phosphate buffer solution (3.2) to
obtain solutions with concentrations corresponding to 100 µg per ml of
chlortetracycline-HCl, of oxytetracycline-HCl and of tetracycline-HCl.
5.3Reference solutions for detection by bioautography
Dilute the solutions (5.1) with the phosphate buffer solution (3.2) to
obtain solutions with concentrations corresponding to 5 µg per ml of
chlortetracycline-HCl, of oxytetracycline-HCl and of tetracycline-HCl.
6.Extraction
When the presumed antibiotic content is less than 10 ppm, either the homo-
genized sample or the finest fraction separated by sieving may be used, since
the antibiotics are to be found mainly in this fraction.
Suspend the sample in the mixture (3.5) and centrifuge. Collect the superna-
tant liquid and use it directly or dilute, if necessary, with the mixture (3.5) to
obtain antibiotic concentrations of approximately 100 µg per ml (6.1) and
5 µg per ml (6.2).
7.Detectionandidentification
7.1Chromatography
Immerse the paper in the buffer solution, pH 3·5 (3.1). Remove the
excess liquid by pressing the paper between sheets of dry filter paper.
Then place volumes of 0·01 ml of the reference solutions (5.2 and 5.3)
and of the extract (6.1 and 6.2) on the paper. To give a good separation,
the paper must have the correct moisture content; if necessary, leave to
dry a little.
Developby ascending chromatograp hy. Use eluent I (3.3) for detection
by bioautography and eluent II (3.4) for detection by UV light. When
the solvent front has climbed 15 to 20 cm (approx. 1 hour 30 minutes),
stop chromatography and dry the paper.
7.2Detection by UV light
If the antibiotic level is greater than 1 µg per cm
2, after the chromato-
gram has been treated with ammonia vapours (3.7) golden yellow
fluorescent spots will be seen on irradiation under the UV lamp (4.4).1972L0199 — EN — 22.07.1993 — 003.001 — 16
▼B
7.3Detection by bioautography
Pour the culture medium (3.10), previously inoculated with B. cereus
(3.9), into glass plates (4.5) and place the paper on the culture medium.
After 5 minutes' contact, detach the paper and place it on another spot in
the culture medium, where it will remain during the incubation period.
The incubate overnight at 30 ºC. If an antibiotic of the tetracycline group
is present, light inhibition zones will appear in the cloudy culture
medium.
To fix the chromatogram, the solution (3.11) is vaporized on the paper,
after incubation.
7.4Identification
The relative Rf values of antibiotics of the tetracycline groupare given
below. These values may vary slightly according to the quality of the
paper and its moisture content:
Chlortetracycline (CTC) 0·60
Tetracycline (TC) 0·40
Oxytetracycline (OTC) 0·20
4-epi-CTC 0·154-epi-TC 0·13
4-epi-OTC 0·10
The antibiotic activity of the ‘epi’ compounds is less than that of the
normal compounds.
2.DETERMINATION OF CHLORTETRACYCLINE, OXYTETRACY-
CLINEANDTETRACYCLINE
A. BY DIFFUSION ON AGAR
1.Purposeandscope
This method makes it possible to determine the levels of chlortetracycline
(CTC), oxytetracycline (OTC) and tetracycline (TC) in feedingstuffs, concen-
trates and premixes where more than 5 ppm are present. Contents of less than
5 ppm may be estimated by graphic interpolation.
2.Principle
For contents of 50 ppm or less, the sample is extracted with dilute forma-
mide. For contents greater than 50 ppm, it is extracted with a mixture of
acetone, water and hydrochloric acid, for the determination of CTC, and
with a mixture of methanol and hydrochloric acid for the determination of
OTC and TC.
The extracts are then diluted and their antibiotic activity determined by
measuring the diffusion of the CTC, OTC or TC on an agar medium seeded
withB. cereus . The diffusion is made evident by the formation of inhibition
zones in the presence of the micro-organism. The diameter of these zones is
directly proportional to the logarithm of the antibiotic concentration.
3.Micro-organism: B.cereus,ATCCNo11.778
3.1Maintenance of the parent strain
Inoculate with B. cereus a tube of sloped agar taken from culture
medium (4.1) free from methylene blue and boric acid . Incubate over-
night at approximately 30ºC. Keep the culture in a refrigerator and re-
inoculate sloped agar with it every 14 days.
3.2Preparation of the spore suspension
Collect the bacteria from a tube of sloped agar (3.1) using 2 to 3 ml of
physiological saline (4.5). With this suspension, seed a Roux flask
containing 300 ml of culture medium (4.1), free from methylene blue
and boric acid, with 3 to 4 % agar concentration. Incubate for 3 to 5
days at 28 to 30 ºC, then collect the spores in 15 ml of ethanol (4.6) after
checking sporulation under a microscope, and homogenize. This suspen-
sion will keepin a refrigerator for 5 months or more.
By preliminary tests on plates with the basic medium for the determina-
tion (4.1), establish the quantity of inoculum which, for the different
concentrations of antibiotic used, will give the largest possible inhibition
zones that are still clear. This quantity is usually between 0·2 and 0·3 ml
per 1000 ml. The culture medium is inoculated at between 50 and 60 ºC.1972L0199 — EN — 22.07.1993 — 003.001 — 17
▼B
4.Culturemediaandreagents
4.1Basic medium for the determination (1)
Glucose 1 g
Tryptic peptone 10 g
Meat extract 1·5 gYeast extract 3 g
Agar, according to quality 10 to 20 g
‘Tween 80’ 1 mlPhosphate buffer solution, pH 5·5 (4.2) 10 ml
5 % (w/v) boric acid solution 15 ml
0·5 % solution of methylene blue ethanol 4 mlDistilled water to 1000 ml
Adjust to pH 5·8 before use.
4.2Phosphate buffer solution, pH 5·5
Potassium dihydrogen phosphate KH
2PO4A.R. 130·86 g
diSodium hydrogen phosphate Na2HPO4·
2H2O A.R. 6·947 g
Distilled water to 1000 ml
4.3 Phosphate buffer solution, pH 5·5, diluted to 1/10.
4.4Phosphate buffer solution, pH 8
Potassium dihydrogen phosphate KH2PO4A.R. 1·407 g
diSodium hydrogen phosphate Na2HPO4·
2H2O A.R. 57·539 g
Distilled water to 1000 ml
4.5 Sterile physiological saline.
4.6 20 % (v/v) ethanol.4.7 Hydrochloric acid 0·1 N.4.8 70 % (v/v) formamide: prepare fresh before use and adjust the pH to
4·5 using sulphuric acid approximately 2 N.
4.9 Mixture of pure acetone/water/hydrochloric acid (d: 1·19): 65/33/2 by
volume.
4.10 Mixture of pure methanol/hydrochloric acid (d: 1·19): 98/2 by volume.4.11 Standard substances: CTC, OTC, TC, the activity of which is expressed
in terms of hydrochloride.
5.Standardsolutions
5.1Chlortetracycline
Using hydrochloric acid (4.7), prepare from the standard solution (4.11)
a stock solution with a concentration corresponding to 500 µg per ml of
chlortetracycline-HCl. This solution will keepfor one week in a refrig-
erator.1972L0199 — EN — 22.07.1993 — 003.001 — 18
(1) Any commercial culture medium of similar composition and giving the same results
may be used.
▼B
From this stock solution, prepare a standard working solution S8with a
concentration corresponding to 0·2 µg per ml of chlortetracycline-HCl.
Dilution is carried out using the phosphate buffer solution, pH 5·5,
diluted to 1/10 (4.3), to which 0.01 % of amido black has been added (1).
Then prepare by successive dilutions (1 + 1), using the buffer solution
(4.3), the following concentrations:
S40·1 µg/ml
S20·05 µg/ml
S10·025 µg/ml
5.2Oxytetracycline
Proceeding as indicated in 5.1, prepare, from a stock solution with a
concentration corresponding to 400 µg per ml of oxytetracycline-HCl, a
standard working solution S8containing 1·6 µg per ml of oxytetracycline-
HCl, and the following concentrations:
S40·8 µg/ml
S20·4 µg/ml
S10·2 µg/ml
5.3Tetracycline
Proceeding as indicated in 5.1, prepare, from a stock solution with a
concentration corresponding to 500 µg per ml of tetracycline-HCl, a stan-
dard working solution S8containing 1·0 µg per ml of tetracycline-HCl
and the following concentrations:
S40·5 µg/ml
S20·25 µg/ml
S10·125 µg/ml
6.Extraction
6.1Contents of 50 ppm or less
To test sample add formamide (4.8) in the quantities indicated in the
table below. Shake for 30 minutes on a shaking platform. Then dilute
immediately with the phosphate buffer solution (4.3) according to the
indications given in the table below to obtain the concentration U8. The
formamide concentration of this solution must not exceed 40 %.
Centrifuge or decant to obtain a clear solution. Then prepare the concen-
trations U4,U2and U1by successive dilutions (1 + 1) using the
phosphate buffer solution (4.3).
Antibiotic CTC OTC TC
Presumed content
in ppm 10 50 10 50 10 50
Test sample in g 10 10 24 9.6 20 10
ml of formamide
(4.8) 100 100 80 100 80 100
ml of phosphate
buffer solution
(4.3)dil.
1: 5
(a)dil.
1: 25
(b)70 200 120dil.
1: 5
(a)
U8concentration
in µg/ml 0·2 0·2 1·6 1·6 1·0 1·0
(a) Take 20 ml of extract and make upto 100 ml in a graduated flask with the
buffer solution.1972L0199 — EN — 22.07.1993 — 003.001 — 19
(1) Amido black is used to make evident the inhibition zones of the standard solutions
(blue rings).
▼B
(b) Take 4 ml of extract and make upto 100 ml in a graduated flask
with the buffer solution.
6.2Contents greater than 50 ppm
6.2.1 Chlortetracycline
To a test sample of 2 to 10 g, depending on the presumed anti-
biotic content of the sample or its manufacturer's guarantee, add
20 times its volume of mixture (4.9). Shake for 30 minutes on a
shaking platform. The pH must remain below 3 during extraction;
if necessary, readjust to pH 3 (using 10 % acetic acid for mineral
compounds). Take an aliquot part of the extract and adjust the pH
to 5·5 using the phosphate buffer solution, pH 8 (4.4) in the
presence of bromocresol green (turning from yellow to blue).
Dilute, using the phosphate buffer solution, pH 5·5, diluted to 1/
10 (4.3), to obtain the concentration U8(see 6.1).
Then prepare the concentrations U4,U2and U1by successive dilu-
tions (1 + 1) using the phosphate buffer solution (4.3).
6.2.2 Oxytetracycline and tetracycline
Proceed as indicated in 6.2.1, using the mixture (4.10) instead of
the mixture (4.9).
7.Determinationmethod
7.1Inoculation of the culture medium
Inoculate at 50 to 60 ºC the basic medium for the determination (4.1)
with the spore suspension (3.2).
7.2Preparation of the trays
Diffusion on agar is carried out in trays using 4 concentrations of the
standard solution (S8,S4,S2,S1) and 4 concentrations of the extract (U8,
U4,U2,U1). The 4 concentrations of extract and of standard solution
must be placed in each tray.
Choose trays, therefore, which are large enough to allow at least 8 holes
10 to 13 mm in diameter to be made in the agar medium. Calculate the
quantity of inoculated culture medium (7.1) needed to provide a uniform
covering approximately 2 mm thick. The test should preferably be
carried out on trays consisting of flat glass plates fitted with a perfectly
level aluminium or plastic ring, 200 mm in diameter and 20 mm high.
Pipette into the holes accurately measured quantities of between 0·10 and
0·15 ml of antibiotic solution, depending on the diameter of the holes.
For each sample, repeat the diffusion at least 4 times with each concen-
tration so that each determination comprises an evaluation of 32
inhibition zones.
7.3Incubation
Incubate the trays for approximately 18 hours at 28 to 30 ºC.
8.Evaluation
Measure the diameter of the inhibition zones, preferably by projection.
Record the measurements on semi-logarithmic paper, plotting the logarithm
of the concentrations against the diameter of theinhibition zones. Trace the
lines of the standard solution and of the extract. Provided there is no interfer-
ence, the two lines will be parallel.
The logarithm of the relative activity is calculated by using the following
formula:
U1þU2þU4/C2U8/C0S1/C0S2/C0S4/C0S8 ðÞ /C1 0/C1602
U4þU8þS4þS8/C0U1/C0U2/C0S1/C0S2
Real activity = presumed activity × relative activity.
9.Repeatability
The difference between the results of two parallel determinations carried out
on the same sample must not exceed 10 %, in relative value.1972L0199 — EN — 22.07.1993 — 003.001 — 20
▼B
B. BY TURBIDIMETRY
1.Purposeandscope
This method makes it possible to determine the levels of chlortetracycline
(CTC), oxytetracycline (OTC) and tetracycline (TC) where concentrations
are greater than 1 g per kg, provided there is no interference from other
substances clouding the extracts. This method is quicker than diffusion on
agar.
2.Principle
For the determination of CTC, the sample is extracted with a mixture of
acetone, water and hydrochloric acid, and with a mixture of methanol and
hydrochloric acid for the determination of OTC and TC.
The extracts are then diluted and their antibiotic effect determined by
measuring the light transmission of a culture medium which has been seeded
withStaphylococcus aureus and to which the antibiotic has been added. The
light transmission depends on the antibiotic concentration.
3.Micro-organism: StaphylococcusaureusK141 (1)
3.1Maintenance of the parent strain
Inoculate with S. aureus a tube of sloped agar taken from the culture
medium (4.1), to which 1·5 to 3 % of agar has been added (depending
on the quality). Incubate overnight at 37 ºC. Keepthe culture in a refrig-
erator and re-inoculate sloped agar with it every 4 weeks. At the same
time prepare sub-cultures for laboratory use.
3.2Preparation of the inoculum
24 hours before use, re-inoculate sloped agar with a sub-culture and
incubate overnight at 37 ºC. Suspend all the culture contained in a tube
of agar in approximately 2 ml of the basic medium (4.1), then transfer
the suspension under sterile conditions into approximately 100 ml of
the same basic medium (4.1). Incubate in a water bath at 37 ºC until
the growth of the strain enters its logarithmic phase (1 hour 30 minutes
to 2 hours).
4.Culturemediaandreagents
4.1Basic medium for the determination (2)
Peptone 5 g
Yeast extract 1·5 g
Meat extract 1·5 gSodium chloride 3·5 g
Glucose 1·0 g
Potassium dihydrogen phosphate KH
2PO4A.R. 1·32 g
diPotassium hydrogen phosphate K2HPO4A.R.3·68 g
Distilled water to 1 000 ml
pH after sterilization: 6·8 to 7·0.
4.2Phosphate buffer solution, pH 4·5
Potassium dihydrogen phosphate KH2PO4A.R. 13·6 g
Distilled water to 1 000 ml
4.3 Hydrochloric acid 0·1 N.
4.4 Mixture of pure acetone/water/hydrochloric acid (d: 1·19): 65/33/2 by
volume.
4.5 Mixture of pure methanol/hydrochloric acid (d: 1·19): 98/2 by volume.1972L0199 — EN — 22.07.1993 — 003.001 — 21
(1) This strain, isolated by the LUFA at Kiel, grows more rapidly than S. aureus ATCC
6538 P.
(2) Any commercial culture medium of similar composition and giving the same results
may be used.
▼B
4.6 Approximately 10 % (w/v) formaldehyde solution.
4.7 Standard substances: CTC, OTC, TC, the activity of which is expressed
in terms of hydrochloride.
5.Standardsolution
Using hydrochloric acid (4.3), prepare from the standard substance (4.7) a
stock solution with a concentration corresponding to 400 to 500 µg per ml
of CTC-HCl, OTC-HCl or TC-HCl. This solution will keepfor one week in
a refrigerator.
6.Extraction
6.1Chlortetracycline
Place a 1 to 2 g test sample in a 200 or 250 ml graduated flask. Add
approximately 100 ml of the mixture (4.4) and shake for 30 minutes on
a shaking platform. Make up to volume with the phosphate buffer solu-
tion, pH 4·5 (4.2). Homogenize and leave to settle.
6.2Oxytetracycline and tetracycline
Place a 1 to 2 g test sample in a 200 or 250 ml graduated flask. Add
approximately 100 ml of the mixture (4.5) and shake for 30 minutes on
a shaking platform. Make up to volume with the phosphate buffer solu-
tion, pH 4·5 (4.2). Homogenize and leave to settle.
7.Determinationmethod
7.1Preparation of the standard series and of the extract
Dilute the standard solution (5) and the extract (6) with the phosphate
buffer solution, pH 4·5 (4.2), to obtain a series of concentrations. For
each determination, a calibration curve is drawn from the respective
concentration, permitting the interpolation of at least two values relating
to the extract. The dilutions should be chosen according to the conditions
under which the strain is grown, which may vary from one laboratory to
another. The procedure is generally the following:
7.1.1 Chlortetracycline
Dilute the standard solution (5) with the phosphate buffer solution
(4.2) to obtain a standard working solution with a concentration
corresponding to 0·2 µg per ml of CTC-HCl. Then, using the phos-
phate buffer solution (4.2), prepare in test tubes, as indicated
below, 6 dilutions, each dilution in duplicate.
ml of standard
working solutionml of phosphate
buffer solution (4.2)Concentration of
CTC-HCl (µg/ml)
0·7 0·3 0·14
0·6 0·4 0·12
0·55 0·45 0·11
0·45 0·55 0·09
0·4 0·6 0·08
0·3 0·7 0·06
Dilute the extract (6.1) with the phosphate buffer solution (4.2) to
obtain a presumed CTC-HCl concentration of 0·12 µg per ml.
Place 1 ml of this solution in each of 2 tubes, and 0·75 ml (—
0·09 µg) in each of 2 other tubes. Make the volume of the latter
two tubes up to 1 ml with the phosphate buffer solution (4.2).
7.1.2 Oxytetracycline and tetracycline
Dilute the standard solution (5) with the phosphate buffer solution
(4.2) to obtain a standard working solution with a concentration
corresponding to 0·6 µg per ml of OTC-HCl or of TC-HCl. Then,
using the phosphate buffer solution (4.2), prepare in test tubes, as
indicated below, 7 dilutions, each dilution in duplicate.1972L0199 — EN — 22.07.1993 — 003.001 — 22
▼B
ml of standard
working solutionml of phosphate
buffer solution (4.2)Concentration of
OTC-HCl or TC-HCl
(µg/ml)
0·9 0·1 0·54
0·8 0·2 0·48
0·7 0·3 0·42
0·6 0·4 0·36
0·4 0·6 0·24
0·3 0·7 0·18
0·2 0·8 0·12
Dilute the extract (6.2) with the phosphate buffer solution (4.2) to
obtain a presumed OTC-HCl or TC-HCl concentration of 0·48 µg
per ml. Place 1 ml of this solution in each of 2 tubes, and 0·5 ml
(= 0·24 µg) in each of 2 other tubes. Makes the volume of the
latter two tubes up to 1 ml with the phosphate buffer solution
(4.2).
7.2Inoculation of the culture medium
Inoculate the basic medium for the determination (4.1) with the
inoculum (3.2) to obtain with the photometer at 590 nm 85 % light trans-
mission in a 5 cm cell or 92 % transmission in a 2 cm cell, the apparatus
being set at 100 % transmission on the non-inoculated basic medium
(4.1).
7.3Seeding
Place 9 ml of the inoculated culture medium (7.2) in each tube (7.1.1 or
7.1.2). The tubes must be filled under clean but not necessarily sterile
conditions.
7.4Incubation
Incubation must be carried out in a water bath whose temperature is kept
uniform at 37 ºC ± 0·1 ºC by stirring. The incubation period chosen
(generally 2 hours 30 minutes to 3 hours) must be such that it will be
possible to trace transmission curves with gradients suitable for accurate
measurement. Then block further growth by rapidly injecting 1 ml of
formaldehyde solution (4.6) into each tube.
7.5Measurement of growth
Measure the transmissions with the photometer at 590 nm, setting the
apparatus at 100 % transmission on the clearest standard solution (corre-
sponding to the highest antibiotic content). Since the different tubes will
show slight differences of turbidity, at least 2 cm, and preferably 5 cm,
cells should be used.
8.Calculationofresults
Trace the calibration curve on millimetre graph paper by plotting the photo-
metric transmissions against the antibiotic concentrations. Interpolate on the
curve the transmission values of the extract. Calculate the antibiotic content
of the sample.
9.Repeatability
The difference between the results of two parallel determinations carried out
on the same sample must not exceed 10 %, in relative value.
3.DETERMINATIONOFOLEANDOMYCIN
— by diffusion on agar —
1.Purposeandscope
This method makes it possible, even in the presence of tetracyclines, to deter-
mine the oleandomycin content of feedingstuffs, concentrates and premixes,
where more than 0.5 ppm are present.1972L0199 — EN — 22.07.1993 — 003.001 — 23
▼B
2.Principle
The sample is extracted with a dilute methanol solution of tri(hydroxymethy-
lamino)methane. After centrifuging, the extract is diluted and its antibiotic
activity determined by measuring the diffusion of the oleandomycin on an
agar medium seeded with B. cereus . The diffusion is made evident by the
formation of inhibition zones in the presence of the micro-organism. The
diameter of these zones is directly proportional to the logarithm of the anti-
biotic concentration.
3.Micro-organism: B.cereusK250TR (1)(resistanttotetracyclines)
3.1Maintenance of the parent strain
Inoculate with B. cereus a tube of sloped agar taken from the culture
medium (4.1) to which 100 µg per 5 ml of oxytetracycline has been
added. Incubate overnight at approximately 30 ºC. Keep the culture in a
refrigerator and re-inoculate sloped agar with it every 4 weeks.
3.2Preparation of the spore suspension
Collect the bacteria from a tube of sloped agar (3.1) using approximately
3 ml of physiological saline (4.3). With this suspension, seed a Roux
flask containing 300 ml of culture medium (4.1) which has a 3 to 4 %
agar concentration . Incubate for 3 to 5 days at 28 to 30 ºC, then collect
the spores in 15 ml of ethanol (4.4) after checking sporulation under a
microscope, and homogenize. This suspension will keep in a refrigerator
for 5 months or more.
By preliminary tests on plates with the basic medium for the determina-
tion (4.2), establish the quantity of inoculum which, for the different
concentrations of oleandomycin used, will give the largest possible inhi-
bition zones that are still clear. This quantity is usually between 0·1 and
0·2 ml per 1000 ml. The culture medium is inoculated at 60 ºC.
4.Culturemediaandreagents
4.1Medium for maintenance of the parent strain (2)
Glucose 1 g
Tryptic peptone 10 g
Meat extract 1·5 g
Yeast extract 3 gAgar, according to quality 10 to 20 g
Distilled water to 1000 ml
Adjust the pH to 6·5 immediately before use.
4.2Basic medium for the determination (
2)
Medium (4.1) adjusted to pH 8·8.
4.3 Sterile physiological saline.4.4 20 % (v/v) ethanol.4.5 Pure methanol.4.6 0·5 % (w/v) solution of tri(hydroxymethylamino)methane A.R.
4.7Extraction solution
Pure methanol 50 ml
Distilled water 50 mlTri(hydroxymethylamino)-
methane A.R.0·5 g
4.8 Standard substance: oleandomycin of known activity.1972L0199 — EN — 22.07.1993 — 003.001 — 24
(1) Strain isolated by the LUFA at Kiel.
(2) Any commercial culture medium of similar composition and giving the same results
may be used.
▼B
5.Standardsolution
Dissolve some of the standard substance (4.8) in 5 ml of methanol (4.5) and
dilute with the solution (4.6) to obtain an oleandomycin concentration of
100 µg per ml.
From this stock solution, prepare a standard working solution S8containing
0.1 µg per ml of oleandomycin by diluting with the solution (4.6). Then
prepare by successive dilutions (1 + 1), using the solution (4.6), the following
concentrations:
S40·05 µg/ml
S20·025 µg/ml
S10·0125 µg/ml
6.Extraction
Take a test sample of 2 to 10 g, depending on the presumed oleandomycin
content of the sample, add 100 ml of the solution (4.7) and shake for 30
minutes on a shaking platform.
Centrifuge, take an aliquot part of the extract and dilute with the solution
(4.6) to obtain a presumed oleandomycin concentration of 0·1 µg per ml (=
U8). Then prepare the concentrations U4,U2and U1by successive dilutions
(1 + 1) using the solution (4.6).
7.Determinationmethod
7.1Inoculation of the culture medium
Inoculate at 60 ºC the basic medium for the determination (4.2) with the
spore suspension (3.2).
7.2Preparation of the trays
Diffusion on agar is carried out in trays using 4 concentrations of the
standard solution (S8,S4,S2,S1) and four concentrations of the extract
(U8,U4,U2,U1). The 4 concentrations of standard solution and of extract
must be placed in each tray.
Choose trays, therefore, which are large enough to allow at least 8 holes
10 to 13 mm in diameter to be made in the agar medium. Calculate the
quantity of inoculated culture medium (7.1) needed to provide a uniform
covering approximately 2 mm thick. The test should preferably be
carried out on trays consisting of flat glass plates fitted with a perfectly
level aluminium or plastic ring 200 mm in diameter and 20 mm high.
Pipette into the holes accurately measured quantities of between 0·10 and
0·15 ml of antibiotic solution, depending on the diameter of the holes.
For each sample repeat the diffusion at least 4 times with each concen-
tration so that each determination comprises an evaluation of 32
inhibition zones.
7.3Incubation
Incubate the trays for approximately 18 hours at 28 to 30 ºC.
8.Evaluation
Measure the diameter of the inhibition zones, preferably by projection.
Record the measurements on semi-logarithmic paper, plotting the logarithm
of the concentrations against the diameter of the inhibition zones. Trace the
lines of the standard solution and of the extract. Provided there is no interfer-
ence, the two lines will be parallel.
The logarithm of the relative activity is calculated by using the following
formula:
U1þU2þU4þU8/C0S1/C0S2/C0S4/C0S8 ðÞ /C1 0/C1602
U4þU8þS4þS8/C0U1/C0U2/C0S1/C0S2
Real activity = presumed activity × relative activity.1972L0199 — EN — 22.07.1993 — 003.001 — 25
▼B
9.Repeatability
The difference between the results of two parallel determinations carried out
on the same sample must not exceed 10% in relative value.
4.DETERMINATIONOFTYLOSIN
— by diffusion on agar —
1.Purposeandscope
This method makes it possible to determine the tylosin content of feeding-
stuffs, concentrates and premixes where more than 2 ppm are present.
2.Principle
The sample is treated with a pH 8 phosphate buffer solution, previously
heated to 80 ºC, and then extracted with methanol. After centrifuging, the
extract is diluted and its antibiotic activity determined by measuring the
diffusion of the tylosin on an agar medium seeded with Sarcina lutea . The
diffusion is made evident by the formation of inhibition zones in the presence
of the micro-organism. The diameter of these zones is directly proportional to
the logarithm of the antibiotic concentration.
3.Micro-organism: SarcinaluteaATCCNo9341
3.1Maintenance of the parent strain
Inoculate with Sarcina lutea a tube of sloped agar taken from the culture
medium (4.1), adjust to pH 7·0. Incubate overnight at approximately
35 ºC. Keepthe culture in a refrigerator and re-inoculate slop ed agar
with it every month.
3.2Preparation of the bacteria suspension
Collect the bacteria from a recently prepared tube of sloped agar (3.1)
using 2 to 3 ml of physiological saline (4.4). With this suspension seed
a Roux flask containing 250 ml of the culture medium (4.1), adjusted to
pH 7·0. Incubate for 24 hours at 35 ºC, then collect the bacteria in 25 ml
of physiological saline (4.4). Homogenize, and dilute this suspension to
obtain approximately 75 % light transmission at 650 nm.
If kept in a refrigerator this suspension may be used for one week.By preliminary tests on plates with the basic medium for the determina-
tion (4.1), establish the quantity of inoculum which, for the different
concentrations of tylosin used, will give the largest possible inhibition
zones that are still clear. The culture medium is inoculated at 48 to 50 ºC.
4.Culturemediaandreagents
4.1Basic medium for the determination (
1)
Glucose 1 g
Tryptic peptone 10 gMeat extract 1·5 g
Yeast extract 3 g
Agar, according to quality 10 to 20 gDistilled water to 1000 ml
Adjust immediately before use to pH 7.0 for maintenance of the parent
strain and preparation of the bacteria suspension, and to pH 8·0 for the
determination.
4.2Phosphate buffer solution, pH 8
Potassium dihydrogen phosphate KH
2PO4A.R. 0·523 g
diPotassium hydrogen phosphate K2HPO4A.R. 16·730 g1972L0199 — EN — 22.07.1993 — 003.001 — 26
(1) Any commercial culture medium of similar composition and giving the same results
may be used
▼B
Distilled water to 1000 ml
4.3Phosphate buffer solution, pH 7
Potassium dihydrogen phosphate KH2PO4A.R. 5·5 g
diPotassium hydrogen phosphate K2HPO4A.R.13·6 g
Distilled water to 1000 ml
4.4 Sterile physiological saline.
4.5 Pure methanol.
4.6 40 % (v/v) methanol.
4.7 Mixture of phosphate buffer solution (4.2)/ pure methanol: 60/40 by
volume.
4.8 Standard substance: tylosin of known activity.
5.Standardsolutions
Dry the standard substance (4.8) for 3 hours at 60 ºC in a vacuum oven
(5 mm of mercury). Weigh 10 to 50 mg into a graduated flask, dissolve in
5 ml of methanol (4.5) and dilute the solution with the the phosphate buffer
solution, pH 7 (4.3), to obtain a tylosin-base concentration of 1000 µg per ml.
Prepare a standard working solution S8containing 2 µg per ml of tylosin base
from this stock solution by diluting with the mixture (4.7).
Then prepare by successive dilutions (1 + 1), using the mixture (4.7), the
following concentrations:
S41 µg/ml
S20·5 µg/ml
S10·25 µg/ml
6.Extraction
For concentrates, take a 10 g test sample; for premixes and feedingstuffs, a
20 g test sample. Add 60 ml of phosphate buffer solution, pH 8 (4.2),
previously heated to 80 ºC, and homogenize for 2 minutes (domestic mixer,
Ultra-turrax, etc.).
Leave to stand for 10 minutes, add 40 ml of methanol (4.5) and homogenize
for 5 minutes. Centrifuge the extract and dilute an aliquot part with the
mixture (4.7) to obtain a presumed tylosin concentration of 2 µg per ml (=
U8). Then prepare the concentrations U4,U2and U1by successive dilutions
(1 + 1) using the mixture (4.7).
For contents of less than 10 ppm, evaporate the extract until dry in a rotary
evaporator at 35 ºC and dissolve the residue in 40 % methanol (4.6).
7.Determinationmethod
7.1Inoculation of the culture medium
Inoculate at 48 to 50 ºC the basic medium for the determination (4.1),
adjust to pH 8·0, with the bacteria suspension (3.2).
7.2Preparation of the trays
Diffusion on agar is carried out in trays using 4 concentrations of the
standard solution (S8,S4,S2,S1) and 4 concentrations of the extract (U8,
U4,U2,U1). The 4 concentrations of standard solution and of extract
must be placed in each tray.
Choose trays, therefore, which are large enough to allow at least 8 holes
10 to 13 mm in diameter to be made in the agar medium. Calculate the
quantity of inoculated culture medium (7.1) needed to provide a uniform
covering approximately 2 mm thick. The test should preferably be
carried out on flat trays consisting of glass plates filled with a perfectly
level aluminium or plastic ring, 200 mm in diameter and 20 mm high.1972L0199 — EN — 22.07.1993 — 003.001 — 27
▼B
Pipette into the holes accurately measured quantities of between 0·10 and
0·15 ml of antibiotic solution, depending on the diameter of the holes.
For each sample repeat the diffusion at least 4 times with each concen-
tration so that each determination comprises an evaluation of 32
inhibition zones.
7.3Incubation
Incubate the trays overnight at 35 to 37 ºC.
8.Evaluation
Measure the diameter of the inhibition zones, preferably by projection.
Record the measurements on semi-logarithmic paper, plotting the logarithm
of the concentrations against the diameter of the inhibition zones. Trace the
lines of the standard solution and of the extract. Provided there is no interfer-
ence the two lines will be parallel.
The logarithm of the relative activity is calculated by using the following
formula:
U1þU2þU4þU8/C0S1/C0S2/C0S4/C0S8 ðÞ /C1 0/C1602
U4þU8þS4þS8/C0U1/C0U2/C0S1/C0S2
Real activity = presumed activity × relative activity.
9.Repeatability
The difference between the results of two parallel determinations carried out
on the same sample must not exceed 10% in relative value.
5.DETERMINATIONOFVIRGINIAMYCIN
— by diffusion in an agar medium —
1.Purposeandscope
The method is for the determination of virginiamycin in feedingstuffs and
premixes. The lower limit of determination is 2 mg/kg (2 ppm)(1).
2.Principle
The sample is extracted with a methanolic solution of Tween 80. The extract
is decanted or centrifuged and diluted. Its antibiotic activity is determined by
measuring the diffusion of virginiamycin in an agar medium inoculated with
Micrococcus luteus. Diffusion is shown by the formation of zones of inhibi-
tion of the micro-organism. The diameter of these zones is taken to be in
direct proportion to the logarithm of the antibiotic concentration over the
range of antibiotic concentrations employed.
3.Micro-organism: Micrococcus luteus ATCC 9341 (NCTC 8340, NCIB
8553)
3.1.Maintenance of stock culture
Inoculate tubes containing slopes of culture medium (4.1) with Micro-
coccus luteus and incubate for 24 hours at 30 ºC. Store the culture in a
refrigerator at about 4 ºC. Reinoculate every two weeks.
3.2.Preparation of the bacterial suspension (a)
Harvest the growth from a recently prepared agar slope (3.1) with 2 to
3 ml of sodium cloride solution (4.3). Use this suspension to inoculate
250 ml of culture medium (4.1) contained in a Roux flask and incubate
for 18 to 20 hours at 30 ºC. Harvest the growth in 25 ml of sodium
chloride solution (4.3) and mix. Dilute the suspension to 1/10 with
sodium chloride solution (4.3). The light transmission of the suspension
must be about 75 %, measured at 650 nm in a 1 cm cell against sodium1972L0199 — EN — 22.07.1993 — 003.001 — 28
(1) 1 mg virginiamycin is equivalent to 1 000 UK units.
(a) Other methods may be used provided that it has been established that they give similar
bacterial suspensions.▼M2
▼M2
chloride solution (4.3). This suspension may be kept for one week at
about 4 ºC.
4.Culturemediaandreagents
4.1.Culture and assay medium (a)
Meat peptone 6,0 g
Tryptone 4,0 g
Yeast extract 3,0 g
Meat extract 1,5 g
Glucose 1,0 g
Agar 10,0 to 20,0 g
Water 1 000 ml
ph 6,5 (after sterilization).
4.2.Phosphate buffer, pH 6
Potassium hydrogen phosphate, K2HPO42,0 g
Potassium dihydrogen phosphate, KH2PO48,0 g
Water to 1 000 ml
4.3. Sodium chloride solution 0,8 % (w/v): dissolve 8 g sodium chloride in
water and dilute to 1 000 ml; sterilize.
4.4. Methanol.
4.5. Mixture of phosphate buffer (4.2)/methanol (4.4): 80/20 (v/v).
4.6. Tween 80 methanolic solution 0,5 % (w/v): dissolve 5 g Tween 80 in
methanol (4.4) and dilute with methanol to 1 000 ml.
4.7. Standard substance: virginiamycin of known activity.
5.Standardsolutions
Dissolve an accurately weighed quantity of the standard substance (4.7) in
methanol (4.4) and dilute with methanol (4.4) to give a stock solution
containing 1 000 µg virginiamycin per ml.
Stored in a stoppered flask at 4 ºC this solution is stable for up to five days.
From this stock solution prepare by successive dilution with the mixture (4.5)
the following solutions:
s81 µg/ml
s40,5 µg/ml
s20,25 µg/ml
s10,125 µg/ml
6.Preparationoftheextractandassaysolutions
6.1.Extraction
6.1.1. Products with a virginiamycin content up to 100 mg/kg
Weigh out a quantity of sample of 50 g, add 200 ml of solution
(4.6) and shake for 30 minutes. Leave to settle or centrifuge, take
20 ml of the supernatant solution and evaporate to about 5 ml in
a rotary evaporator at a temperature not exceeding 40 ºC. Dilute
the residue with the mixture (4.5) to obtain an expected virginia-
mycin content of 1 µg/ml (= u8).
6.1.2. Products with a virginiamycin content greater than 100 mg/kg
Weigh out a quantity of sample not exceeding 10,0 g and
containing between 1 and 50 mg virginiamycin, add 100 ml of
solution (4.6) and shake for 30 minutes. Leave to settle or centri-
fuge, then dilute the supernatant solution with the mixture (4.5) to
obtain an expected virginiamycin content of 1 µg/ml (= u8).1972L0199 — EN — 22.07.1993 — 003.001 — 29
(a) Any commercial culture medium of similar composition and giving the same results
may be used.
▼M2
6.2.Assay solutions
From solution u8prepare solutions u4(expected content: 0,5 µg/ml), u2(expected content: 0,25 µg/ml) and u1(expected content: 0,125 µg/ml)
by means of successive dilution (1 + 1) with the mixture (4.5).
7.Assayprocedure
7.1.Inoculation of the assay medium
Inoculate the assay medium (4.1) with the bacterial suspension (3.2) at
about 50 ºC. By preliminary trials on plates with the medium (4.1)
determine the quantity of bacterial suspension required to give the
largest and clearest zones of inhibition with the various concentrations
of virginiamycin.
7.2.Preparation of the plates
Diffusion through agar is carried out in plates with the four concentra-
tions of the standard solution (s8,s4,s2and s1) and the four
concentrations of the assay solution (u8,u4,u2and u1). These four
concentrations of extract and standard must necessarily be placed in
each plate. To this effect, select plates big enough to allow at least eight
holes with a diameter of 10 to 13 mm and not less than 30 mm between
centres to be made in the agar medium. The test may be carried out on
plates consisting of a sheet of glass with a faced aluminium or plastic
ring placed on top, 200 mm in diameter and 20 mm high.
Pour into the plates a quantity of the medium (4.1) inoculated as in
point 7.1, to give a layer about 2 mm thick (60 ml for a plate of
200 mm diameter). Allow to set in a level position, bore the holes and
place in them exactly measured volumes of assay and standard solutions
(between 0,10 and 0,15 ml per hole, according to the diameter). Apply
each concentration at least four times so that each determination is
subject to an evaluation of 32 zones of inhibition.
7.3.Incubation
Incubate the plates for 16 to 18 hours at 30 ± 2 ºC.
8.Evaluation
Measure the diameter of the zones of inhibition to the nearest 0,1 mm.
Record the mean measurements for each concentration on semi-logarithmic
graph paper showing the logarithm of the concentrations in relation to the
diameters of the zones of inhibition. Plot the best fit lines of both the stan-
dard solution and the extract, for example as below:
Determine the ‘best fit’ point for the standard lowest level (SL) using the
formula:
(a) SL ¼7s1þ4s2þs4/C02s8
10
Determine the ‘best fit’ point for the standard highest level (SH) using the
formula:
(b) SH ¼7s8þ4s4þs2/C02s1
10
Similarly calculate the ‘best fit’ points for the extract lowest level (UL) and
the extract highest level (UH) by substituting u1,u2,u4and u8for s1,s2,s4and
s8in the above formulae.
Record the calculated SL and SH values on the same graph paper and join
them to give the ‘best fit’ line for the standard solution. Similarly record
UL and UH and join them to give the ‘best fit’ line for the extract.
In the absence of any interference the lines should be parallel. For practical
purposes the lines can be considered parallel if the values (SH—SL) and
(UH—UL) do not differ by more than 10 % from their mean value.1972L0199 — EN — 22.07.1993 — 003.001 — 30
▼M2
If the lines are found to be non-parallel either u1and s1or u8and s8may be
discarded and SL, SH, UL and UH calculated, using the alternative formulae,
to give alternative ‘best fit’ lines:
(a′)S L=5s1þ2s2/C0s4
6or5s2þ2s4/C0s8
6
(b′)S H=5s4þ2s2/C0s1
6or5s8þ2s4/C0s2
6
and similarly for UL and UH. The same criteria of parallelism should be
satisfied. The fact that the result has been calculated from three levels should
be noted on the final report.
When the lines are considered as being parallel, calculate the logarithm of
the relative activity (log A) by means of one of the following formulae,
depending upon whether three or four levels have been used for the assess-
ment of parallelism.
For four levels
(c) Log A ¼u1þu2þu4þu8/C0s1/C0s2/C0s4/C0s8 ðÞ /C2 0;602
u4þu8þs4þs8/C0u1/C0u2/C0s1/C0s2
For three levels
(d) Log A ¼u1þu2þu4/C0s1/C0s2/C0s4 ðÞ /C2 0;401
u4þs4/C0u1/C0s1
or
(d′) Log A ¼u2þu4þu8/C0s2/C0s4/C0s8 ðÞ /C2 0;401
u8þs8/C0u2/C0s2
Activity of sample extract = activity of relevant standard × A
(u8=s8×A )
If the relative activity is found to be outside the range of 0,5 to 2,0, then
repeat the assay making appropriate adjustments to the extract concentrations
or, if this is not possible, to the standard solutions. When the relative activitycannot be brought into the required range, any result obtained must be
considered as approximate and this should be noted on the final report.
When the lines are considered as not being parallel, repeat the determination.
If parallelism is still not achieved, the determination must be considered as
unsatisfactory.
Express the result in milligrams of virginiamycin per kilogram of feeding-
stuff.
9.Repeatability
The difference between the results of two parallel determinations carried out
on the same sample by the same analyst should not exceed:
— 2 mg/kg, in absolute value, for contents of virginiamycin upto 10 mg/kg,
— 20 % related to the highest value for contents of 10 to 25 mg/kg,— 5 mg/kg, in absolute value, for contents of 25 to 50 mg/kg,— 10 % related to the highest value for contents above 50 mg/kg.1972L0199 — EN — 22.07.1993 — 003.001 — 31