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# 31984L0004

**Commission Directive 84/4/EEC of 20 December 1983 amending Directives 71/393/EEC, 72/199/EEC and 78/633/EEC establishing Community methods of analysis for the official control of feedingstuffs **

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Official Journal L 015 , 18/01/1984 P. 0028 - 0038 Finnish special edition: Chapter 3 Volume 17 P. 0033 Spanish special edition: Chapter 03 Volume 29 P. 0233 Swedish special edition: Chapter 3 Volume 17 P. 0033 Portuguese special edition Chapter 03 Volume 29 P. 0233 *

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COMMISSION DIRECTIVE

of 20 December 1983

amending Directives 71/393/EEC, 72/199/EEC and 78/633/EEC establishing Community methods of analysis for the official control of feedingstuffs

(84/4/EEC)

THE COMMISSION OF THE EUROPEAN

COMMUNITIES,

Having regard to the Treaty establishing the European Economic Community,

Having regard to Council Directive 70/373/EEC of 20 July 1970 on the introduction of Community methods of sampling and analysis for the official control of feedingstuffs (1), as last amended by the Act of Accession of Greece, and in particular Article 2 thereof,

Whereas Commission Directives 71/393/EEC (2), 72/199/EEC (3) and 78/633/EEC (4) set out the methods of analysis for crude oils and fats, virginiamycin and zinc bacitracin; whereas there is a need to replace these methods by methods which reflect advances in scientific and technical knowledge;

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

In the Annex to Directive 71/393/EEC, part IV 'Determination of crude oils and fats' is replaced by Annex I to this Directive.

Article 2

In Annex II to Directive 72/199/EEC, part 5 'Determination of virginiamycin by diffusion in an agar medium' is replaced by Annex II to this Directive.

Article 3

In the Annex to Directive 78/633/EEC, part 1 'Determination of zinc bacitracin by diffusion in an agar medium' is replaced by Annex III to this Directive.

Article 4

Member States shall, by 1 June 1984 at the latest, bring into force the laws, regulations or administrative provisions necessary to comply with this Directive and shall forthwith inform the Commission thereof.

Article 5

This Directive is addressed to the Member States.

Done at Brussels, 20 December 1983.

For the Commission

Poul DALSAGER

Member of the Commission

(1) OJ No L 170, 3. 8. 1970, p. 2.

(2) OJ No L 279, 20. 12. 1971, p. 7.

(3) OJ No L 123, 29. 5. 1972, p. 6.

(4) OJ No L 206, 29. 7. 1978, p. 43.

ANNEX I

'4. DETERMINATION OF CRUDE OILS AND FATS

1. Purpose and scope

This method makes it possible to determine the content of crude oils and fats in feedingstuffs. It does not cover the analysis of the oil seeds and oleaginous fruit defined in Council Regulation 136/66/EEC of 22 September 1966.

Depending on the nature of the feedingstuff, either of the two methods described must be used.

1.1. Method A

Applicable to sraight feedingstuffs of plant origin, with the exception of those which are known to contain oils and fats which cannot be totally extracted with light petroleum without prior hydrolysis. Among these are glutens, yeasts, soya and potato proteins. This method is also applicable to compound feedingstuffs, with the exception of those which contain milk powder or from which oils and fats cannot be totally extracted with light petroleum without prior hydrolysis.

1.2. Method B

Applicable to straight feedingstuffs of animal origin as well as to feedingstuffs mentioned under point 1.1 as being exceptions for method A.

2. Principle

2.1. Method A

The sample is extracted with light petroleum. The solvent is distilled off and the residue dried and weighed.

2.2. Method B

The sample is treated under heating with hydrochloric acid. The mixture is cooled and filtered. The residue is washed and dried and submitted to the determination according to method A.

3. Reagents

3.1. Light petroleum, boiling range: 40 to 60 °C. The bromine value must be less than 1 and the residue of evaporation less than 2 mg/100 ml.

3.2. Sodium sulphate, anhydrous.

3.3. Hydrochloric acid 3N.

3.4. Filtration aid, e.g. Kieselgur, Hyflo-supercel.

4. Apparatus

4.1. Extraction apparatus. If fitted with a siphon (Soxhlet apparatus), the reflux rate should be such as to produce about 10 cycles per hour; if of the non-siphoning type, the reflux rate should be about 10 ml per minute.

4.2. Extraction thimbles, free of matter soluble in light petroleum and having a porosity consistent with the requirements of point 4.1.

4.3. Drying oven, either a vacuum oven set at 75 ± 3 °C or an air-oven set at 100 ± 3 °C.

5. Procedure

5.1. Method A (see point 8.1)

Weigh 5 g of the sample to the nearest 1 mg, transfer it to an extraction thimble (4.2) and cover with a fat-free wad of cotton wool.

Place the thimble in an extractor (4.1) and extract for six hours with light petroleum (3.1). Collect the light petroleum extract in a dry, weighed flask containing fragments of pumice stone (1).

Distil off the solvent. Dry the evaporation residue maintaining the flask for one and a half hours in the drying oven (4.3). Leave to cool in a dessiccator and weigh. Dry again for 30 minutes to ensure that the weight of the oils and fats remains constant (loss in weight between two successive weighings must be less than 1 mg).

5.2. Method B

Weigh 2,5 g of the sample to the nearest 1 mg (see point 8.2), place in a 400 ml beaker or a 300 ml conical flask and add 100 ml of hydrochloric acid 3N (3.3) and fragments of pumice stone. Cover the beaker with a watch glass or fit the conical flask with a reflux condenser. Bring the mixture to a gentle boil over a low flame or a hot-plate and keep it there for one hour. Do not allow the product to stick to the sides of the container.

Cool and add a quantity of filtration aid (3.4) sufficient to prevent any loss of oil and fat during filtration. Filter through a moistened, fat-free, double filter paper. Wash the residue in cold water until a neutral filtrate is obtained. Check that the filtrate does not contain any oil or fats. Their presence indicates that the sample must be extracted with light petroleum, using method A, before hydrolysis.

Place the double filter paper containing the residue on a watch glass and dry for one and a half hours in the oven at 100 ± 3 °C.

Place the double filter paper containing the dry residue in an extraction thimble (4.2) and cover with a fat-free wad of cotton wool. Place the thimble in an extractor (4.1) and proceed as indicated in the second and third paragraphs of point 5.1.

6. Expression of result

Express the weight of the residue as a percentage of the sample.

7. Repeatability

The difference between the results of two parallel determinations carried out on the same sample by the same analyst should not exceed:

- 0,2 %, in absolute value, for contents of crude oils and fats lower than 5 %,

- 4,0 % related to the highest result for contents of 5 to 10 %,

- 0,4 % in absolute value, for contents above 10 %.

8. Observations

8.1. For products with a high content of oils and fats, which are difficult to crush or unsuitable for drawing a homogeneous reduced test sample, proceed as follows.

Weigh 20 g of the sample to the nearest 1 mg and mix with 10 g or more of anhydrous sodium sulphate (3.2). Extract with light petroleum (3.1) as indicated in point 5.1. Make up the extract obtained to 500 ml with light petroleum (3.1) and homogenize. Take 50 ml of the solution and place in a small, dry, weighed flask containing fragments of pumice stone (1). Distil off the solvent, dry and proceed as indicated in the last paragraph of point 5.1.

Eliminate the solvent from the extraction residue left in the thimble, crush the residue to a fineness of 1 mm, return it to the extraction thimble (do not add sodium sulphate) and proceed as indicated in the second and third paragraphs of point 5.1.

Calculate the content of oils and fats as a percentage of the sample by using the following formula:

(10 a + b) × 5

where:

a = mass in grams of the residue after the first extraction (aliquot part of the extract),

b = mass in grams of the residue after the second extraction.

8.2. For products low in oils and fats the test sample may be increased to 5 g.'

(1) Where the oil or fat has to undergo subsequent quality tests, replace the fragments of pumice stone by glass beads.

(1) Where the oil or fat has to undergo subsequent quality tests, replace the fragments of pumice stone by glass beads.

ANNEX II

'5. Determination of virginiamycin

- by diffusion in an agar medium -

1. Purpose and scope

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 inhibition 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 Micrococcus luteus and incubate for 24 hours at 30 °C. Store the culture in a refrigerator at about 4 °C. 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 °C. 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 sodium chloride solution (4.3). This suspension may be kept for one week at about 4 °C.

4. Culture media and reagents

4.1. Culture and assay medium (b)

1.2 // 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

1.2 // Potassium hydrogen phosphate, K2HPO4 // 2,0 g // Potassium dihydrogen phosphate, KH2PO4 // 8,0 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. Standard solutions

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 mg virginiamycin per ml.

Stored in a stoppered flask at 4 °C 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:

s8 1 mg/ml

s4 0,5 mg/ml

s2 0,25 mg/ml

s1 0,125 mg/ml

6. Preparation of the extract and assay solutions

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 °C. Dilute the residue with the mixture (4.5) to obtain an expected virginiamycin content of 1 mg/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 centrifuge, then dilute the supernatant solution with the mixture (4.5) to obtain an expected virginiamycin content of 1 m g/ml (= u8).

6.2. Assay solutions

From solution u8 prepare solutions u4 (expected content: 0,5 m g/ml), u2 (expected content: 0,25 mg/ml) and u1 (expected content: 0,125 mg/ml) by means of successive dilution (1 + 1) with the mixture (4.5).

7. Assay procedure

7.1. Inoculation of the assay medium

Inoculate the assay medium (4.1) with the bacterial suspension (3.2) at about 50 °C. 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 concentrations of the standard solution (s8, s4, s2 and s1) and the four concentrations of the assay solution (u8, u4, u2 and 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 °C.

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 standard solution and the extract, for example as below: Determine the "best fit" point for the standard lowest level (SL) using the formula:

1.2 // (a) SL = // 7s1 + 4s2 + s4 - 2s8 10

Determine the ''best fit" point for the standard highest level (SH) using the formula:

1.2 // (b) SH = // 7s8 + 4s4 + s2 - 2s1 10

Similarly calculate the "best fit" points for the extract lowest level (UL) and the extract highest level (UH) by substituting u1, u2, u4 and u8 for s1, s2, s4 and s8 in 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.

If the lines are found to be non-parallel either u1 and s1 or u8 and s8 may be discarded and SL, SH, UL and UH calculated, using the alternative formulae, to give alternative "best fit" lines:

1.2.3.4 // (a' ) SL = // 5s1 + 2s2 - s4 6 // or // 5s2 + 2s4 - s8 6 // (b' ) SH = // 5s4 + 2s2 - s1 6 // or // 5s8 + 2s4 - s2 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 assessment of parallelism.

For four levels

1.2 // (c) Log A = // (u1 + u2 + u4 + u8 - s1 - s2 - s4 - s8) × 0,602 u4 + u8 + s4 + s8 - u1 - u2 - s1 - s2

For three levels

1.2 // (d) Log A = // (u1 + u2 + u4 - s1 - s2 - s4) × 0,401 u4 + s4 - u1 - s1 // or // // (d' ) Log A = // (u2 + u4 + u8 - s2 - s4 - s8) × 0,401 u8 + s8 - u2 - s2

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 activity cannot 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 feedingstuff. 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 up to 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.'

(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.

(b) Any commercial culture medium of similar composition and giving the same results may be used.

ANNEX III

'1. DETERMINATION OF ZINC BACITRACIN

- by diffusion in an agar medium -

1. Purpose and scope

The method is for the determination of zinc bacitracin in feedingstuffs and premixes. The lower limit of determination is 5 mg/kg (5 ppm) (1).

2. Principle

The sample is extracted at pH 2 with a mixture of methanol/water/hydrochloric acid, and a sodium sulphide solution. The addition of sodium sulphide is to precipitate any soluble copper salts that may interfere with the assay. The extract is brought to pH 6,5, concentrated (where necessary) and diluted. Its antibiotic activity is determined by measuring the diffusion of zinc bacitracin in an agar medium inoculated with Micrococcus luteus (flavus). Diffusion is shown by the formation of zones of inhibition 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 (flavus) ATCC 10240

3.1. Maintenance of stock culture

Inoculate tubes containing slopes of culture medium (4.1) with Micrococcus luteus (flavus) and incubate for 24 hours at 30 °C. Store the culture in a refrigerator at about 4 °C. 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 chloride 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 °C. 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 sodium chloride solution (4.3). This suspension may be kept for one week at about 4 °C.

4. Culture media and reagents

4.1. Culture medium (b)

1.2 // 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 to 6,6 (after sterilization). //

4.2. Assay medium (b)

1.2 // Tryptone // 10,0 g // Yeast extract // 3,0 g // Meat extract // 1,5 g // Glucose // 1,0 g // Agar // 10,0 to 20,0 g // Tween 80 // 1 ml // Water // 1 000 ml // pH 6,5 (after sterilization). //

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. Mixture of methanol/water hydrochloric acid (4.6):

80/17,5/2,5 (v/v/v).

4.5. Phosphate buffer, pH 6,5:

1.2 // Potassium hydrogen phosphate K2HPO4 // 22,15 g. // Potassium dihydrogen phosphate KH2PO4 // 27,85 g. // Water to // 1 000 ml.

4.6. Hydrochloric acid (d: 1,18 to 1,19).

4.7. Hydrochloric acid (0,1 M).

4.8. Sodium hydroxide 1 M solution.

4.9. Sodium sulphide about 0,5 M solution.

4.10. Bromocresol purple solution 0,04 % (w/v): dissolve 0,1 g of bromocresol purple in 18,5 ml of 0,01 M sodium hydroxide solution. Make up the volume to 250 ml with water and mix.

4.11. Standard substance: zinc bacitracin of known activity (in i. u.).

5. Standard solutions

Weigh out a quantity of standard zinc bacitracin (4.11) corresponding to 1 050 i.u. (according to the activity indicated). Add 5 ml of 0,1 M hydrochloric acid (4.7) and leave to stand for 15 minutes. Add 30 ml of water, adjust the pH to 4,5 with phosphate buffer (4.5) (about 4 ml), make up to a volume of 50 ml with water and mix well (1 ml = 21 i.u.).

From this solution prepare by successive dilution with phosphate buffer (4.5) the following solutions:

s8 0,42 i.u./ml

s4 0,21 i.u./ml

s2 0,105 i.u./ml

s1 0,0525 i.u./ml

6. Preparation of the extract and assay solutions

6.1. Extraction

6.1.1. Premixes and mineral feeds

Weigh out a quantity of sample of 2,0 to 5,0 g, add 29,0 ml of the mixture (4.4) and 1,0 ml of sodium sulphide solution (4.9) and shake briefly. Check that the pH is about 2. Shake for 10 minutes, add 30 ml of phosphate buffer (4.5), shake for 15 minutes and centrifuge. Take a suitable aliquot of the supernatant solution and adjust the pH to 6,5 by means of 1 M hydroxide solution (4.8) with a pH-meter or with the bromocresol purple solution (4.10) as indicator. Dilute with phosphate buffer (4.5) to obtain an expected zinc bacitracin content of 0,42 i.u./ml (=u8).

6.1.2. Protein concentrates

Weigh out a quantity of sample of 10,0 g, add 49,0 ml of the mixture (4.4) and 1,0 ml of sodium sulphide solution (4.9) and shake briefly. Check that the pH is about 2. Shake for 10 minutes. Add 50 ml of phosphate buffer (4.5), shake for 15 minutes and centrifuge. Take a suitable volume of the supernatant solution and adjust the pH to 6,5 by means of 1 M sodium hydroxide solution (4.8) with a pH-meter or with the bromocresol purple solution (4.10) as indicator. Evaporate to approximately half volume in a rotary evaporator at a temperature not exceeding 35 °C.

Dilute with phosphate buffer (4.5) to obtain an expected zinc bacitracin content of 0,42 i.u./ml (= u8).

6.1.3. Other feeds

Weigh out a quantity of sample of 10,0 g (20,0 g for an expected zinc bacitracin content of 5 mg/kg). Add a mixture of 24,0 ml of the mixture (4.4) and 1,0 ml of sodium sulphide solution (4.9) and homogenize for 10 minutes. Add 25 ml of phosphate buffer (4,5), shake for 15 minutes and centrifuge. Take 20 ml of the supernatant solution and adjust the pH to 6.5 by means of 1 M sodium hydroxide solution (4.8) with a pH-meter or with the bromocresol purple solution (4.10) as indicator. Evaporate to about 4 ml in a rotary evaporator at a temperature not exceeding 35 °C. Dilute the residue with phosphate buffer (4.5) to obtain an expected zinc bacitracin content of 0,42 i.u./ml (= u8).

6.2. Assay solutions

From solution u8 prepare solutions u4 (expected content: 0,21 i.u./ml), u2 (expected content: 0,105 i.u./ml) and u1 (expected content: 0,0525 i.u./ml) by means of successive dilution (1 + 1) with phosphate buffer (4.5). 7. Assay procedure

7.1. Inoculation of the assay medium

Inoculate the assay medium (4.2) with the bacterial suspension (3.2) at about 50 °C. By preliminary trials on plates with assay medium (4.2) determine the quantity of bacterial suspension required to give the largest and clearest zones of inhibition with the various concentrations of zinc bacitracin.

7.2. Preparation of the plates

Diffusion through agar is carried out in plates with the four concentrations of the standard solution (s8, s4, s2 and s1) and the four concentrations of the assay solution (u8, u4, u2 and 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 (4.2) 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 °C.

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 standard solution and the extract, for example as below:

Determine the "best fit" point for the standard lowest level (SL) using the formula:

1.2 // (a) SL = // 7s1 + 4s2 + s4 - 2s8 10

Determine the ''best fit" point for the standard highest level (SH) using the formula:

1.2 // (b) SH = // 7s8 + 4s4 + s2 - 2s1 10

Similarly calculate the "best fit" points for the extract lowest level (UL) and the extract highest level (UH) by substituting u1, u2, u4 and u8 for s1, s2, s4 and s8 in 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.

If the lines are found to be non-parallel either u1 and s1 or u8 and s8 may be discarded and SL, SH, UL and UH calculated, using the alternative formulae, to give alternative "best fit" lines:

1.2.3.4 // (a' ) SL = // 5s1 + 2s2 - s4 6 // or // 5s2 + 2s4 - s8 6 // (b' ) SH = // 5s4 + 2s2 - s1 6 // or // 5s8 + 2s4 - s2 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 (log A) of the relative activity (A) by means of one of the following formulae, depending upon whether three or four levels have been used for the assessment of parallelism.

For four levels

1.2 // (c) log A = // (u1 + u2 + u4 + u8 - s1 - s2 - s4 - s8) × 0,602 u4 + u8 + s4 + s8 - u1 - u2 - s1 - s2

For three levels

1.2 // (d) log A = // (u1 + u2 + u4 - s1 - s2 - s4) × 0,401 u4 + s4 - u1 - s1 // or // // (d' ) log A = // (u2 + u4 + u8 - s2 - s4 - s8) × 0,401 u8 + s8 - u2 - s2

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 activity cannot 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, a satisfactory determination has not been obtained.

Express the result in milligrams of zinc bacitracin per kilogram of feedingstuff.

9. Repeatability

The difference between the results of two determinations carried out on the same sample by the same analyst should not exceed:

- 2 mg/kg, in absolute value, for contents of zinc bacitracin up to 10 mg/kg,

- 20 % related to the highest value for contents from 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.'

(1) 1 mg feedingstuff grade zinc bacitracin is equivalent to 42 international units (i.u.).

(a) Other methods may be used provided that it has been established that they give similar bacterial suspensions.

(b) Any commercial culture medium of similar composition and giving the same results may be used.

4.11 . STANDARD SUBSTANCE : ZINC BACITRACIN OF KNOWN ACTIVITY ( IN I . U .).

5 . STANDARD SOLUTIONS

WEIGH OUT A QUANTITY OF STANDARD ZINC BACITRACIN ( 4.11 ) CORRESPONDING TO 1 050 I.U . ( ACCORDING TO THE ACTIVITY INDICATED ). ADD 5 ML OF 0,1 M HYDROCHLORIC ACID ( 4.7 ) AND LEAVE TO STAND FOR 15 MINUTES . ADD 30 ML OF WATER, ADJUST THE PH TO 4,5 WITH PHOSPHATE BUFFER ( 4.5 ) ( ABOUT 4 ML ), MAKE UP TO A VOLUME OF 50 ML WITH WATER AND MIX WELL ( 1 ML = 21 I.U .).

FROM THIS SOLUTION PREPARE BY SUCCESSIVE DILUTION WITH PHOSPHATE BUFFER ( 4.5 ) THE FOLLOWING SOLUTIONS :

S8 0,42 I.U./ML

S4 0,21 I.U./ML

S2 0,105 I.U./ML

S1 0,0525 I.U./ML

6 . PREPARATION OF THE EXTRACT AND ASSAY SOLUTIONS

6.1 . EXTRACTION

6.1.1 . PREMIXES AND MINERAL FEEDS

WEIGH OUT A QUANTITY OF SAMPLE OF 2,0 TO 5,0 G, ADD 29,0 ML OF THE MIXTURE ( 4.4 ) AND 1,0 ML OF SODIUM SULPHIDE SOLUTION ( 4.9 ) AND SHAKE BRIEFLY . CHECK THAT THE PH IS ABOUT 2 . SHAKE FOR 10 MINUTES, ADD 30 ML OF PHOSPHATE BUFFER ( 4.5 ), SHAKE FOR 15 MINUTES AND CENTRIFUGE . TAKE A SUITABLE ALIQUOT OF THE SUPERNATANT SOLUTION AND ADJUST THE PH TO 6,5 BY MEANS OF 1 M HYDROXIDE SOLUTION ( 4.8 ) WITH A PH-METER OR WITH THE BROMOCRESOL PURPLE SOLUTION ( 4.10 ) AS INDICATOR . DILUTE WITH PHOSPHATE BUFFER ( 4.5 ) TO OBTAIN AN EXPECTED ZINC BACITRACIN CONTENT OF 0,42 I.U./ML (= U8 ).

6.1.2 . PROTEIN CONCENTRATES

WEIGH OUT A QUANTITY OF SAMPLE OF 10,0 G, ADD 49,0 ML OF THE MIXTURE ( 4.4 ) AND 1,0 ML OF SODIUM SULPHIDE SOLUTION ( 4.9 ) AND SHAKE BRIEFLY . CHECK THAT THE PH IS ABOUT 2 . SHAKE FOR 10 MINUTES . ADD 50 ML OF PHOSPHATE BUFFER ( 4.5 ), SHAKE FOR 15 MINUTES AND CENTRIFUGE . TAKE A SUITABLE VOLUME OF THE SUPERNATANT SOLUTION AND ADJUST THE PH TO 6,5 BY MEANS OF 1 M SODIUM HYDROXIDE SOLUTION ( 4.8 ) WITH A PH-METER OR WITH THE BROMOCRESOL PURPLE SOLUTION ( 4.10 ) AS INDICATOR . EVAPORATE TO APPROXIMATELY HALF VOLUME IN A ROTARY EVAPORATOR AT A TEMPERATURE NOT EXCEEDING 35 C .

DILUTE WITH PHOSPHATE BUFFER ( 4.5 ) TO OBTAIN AN EXPECTED ZINC BACITRACIN CONTENT OF 0,42 I.U./ML (= U8 ).

6.1.3 . OTHER FEEDS

WEIGH OUT A QUANTITY OF SAMPLE OF 10,0 G ( 20,0 G FOR AN EXPECTED ZINC BACITRACIN CONTENT OF 5 MG/KG ). ADD A MIXTURE OF 24,0 ML OF THE MIXTURE ( 4.4 ) AND 1,0 ML OF SODIUM SULPHIDE SOLUTION ( 4.9 ) AND HOMOGENIZE FOR 10 MINUTES . ADD 25 ML OF PHOSPHATE BUFFER ( 4,5 ), SHAKE FOR 15 MINUTES AND CENTRIFUGE . TAKE 20 ML OF THE SUPERNATANT SOLUTION AND ADJUST THE PH TO 6.5 BY MEANS OF 1 M SODIUM HYDROXIDE SOLUTION ( 4.8 ) WITH A PH-METER OR WITH THE BROMOCRESOL PURPLE SOLUTION ( 4.10 ) AS INDICATOR . EVAPORATE TO ABOUT 4 ML IN A ROTARY EVAPORATOR AT A TEMPERATURE NOT EXCEEDING 35 C . DILUTE THE RESIDUE WITH PHOSPHATE BUFFER ( 4.5 ) TO OBTAIN AN EXPECTED ZINC BACITRACIN CONTENT OF 0,42 I.U./ML (= U8 ).

6.2 . ASSAY SOLUTIONS

FROM SOLUTION U8 PREPARE SOLUTIONS U4 ( EXPECTED CONTENT : 0,21 I.U./ML ), U2 ( EXPECTED CONTENT : 0,105 I.U./ML ) AND U1 ( EXPECTED CONTENT : 0,0525 I.U./ML ) BY MEANS OF SUCCESSIVE DILUTION ( 1 + 1 ) WITH PHOSPHATE BUFFER ( 4.5 ).

7 . ASSAY PROCEDURE

7.1 . INOCULATION OF THE ASSAY MEDIUM

INOCULATE THE ASSAY MEDIUM ( 4.2 ) WITH THE BACTERIAL SUSPENSION ( 3.2 ) AT ABOUT 50 C . BY PRELIMINARY TRIALS ON PLATES WITH ASSAY MEDIUM ( 4.2 ) DETERMINE THE QUANTITY OF BACTERIAL SUSPENSION REQUIRED TO GIVE THE LARGEST AND CLEAREST ZONES OF INHIBITION WITH THE VARIOUS CONCENTRATIONS OF ZINC BACITRACIN .

7.2 . PREPARATION OF THE PLATES

DIFFUSION THROUGH AGAR IS CARRIED OUT IN PLATES WITH THE FOUR CONCENTRATIONS OF THE STANDARD SOLUTION ( S8, S4, S2 AND S1 ) AND THE FOUR CONCENTRATIONS OF THE ASSAY SOLUTION ( U8, U4, U2 AND 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 ( 4.2 ) 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 C .

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 STANDARD SOLUTION AND THE EXTRACT, FOR EXAMPLE AS BELOW :

DETERMINE THE BEST FIT" POINT FOR THE STANDARD LOWEST LEVEL ( SL ) USING THE FORMULA :

1.2(A ) SL =

7S1 + 4S2 + S4 - 2S8 10

DETERMINE THE BEST FIT" POINT FOR THE STANDARD HIGHEST LEVEL ( SH ) USING THE FORMULA :

1.2(B ) SH =

7S8 + 4S4 + S2 - 2S1 10

SIMILARLY CALCULATE THE BEST FIT" POINTS FOR THE EXTRACT LOWEST LEVEL ( UL ) AND THE EXTRACT HIGHEST LEVEL ( UH ) BY SUBSTITUTING U1, U2, U4 AND U8 FOR S1, S2, S4 AND S8 IN 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 .

IF THE LINES ARE FOUND TO BE NON-PARALLEL EITHER U1 AND S1 OR U8 AND S8 MAY BE DISCARDED AND SL, SH, UL AND UH CALCULATED, USING THE ALTERNATIVE FORMULAE, TO GIVE ALTERNATIVE BEST FIT" LINES :

1.2.3.4(A' ) SL =

5S1 + 2S2 - S4 6

OR

5S2 + 2S4 - S8 6

( B' ) SH =

5S4 + 2S2 - S1 6

OR

5S8 + 2S4 - S2 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 ( LOG A ) OF THE RELATIVE ACTIVITY ( A ) BY MEANS OF ONE OF THE FOLLOWING FORMULAE, DEPENDING UPON WHETHER THREE OR FOUR LEVELS HAVE BEEN USED FOR THE ASSESSMENT OF PARALLELISM .

FOR FOUR LEVELS

1.2(C ) LOG A =

( U1 + U2 + U4 + U8 - S1 - S2 - S4 - S8 ) 0,602 U4 + U8 + S4 + S8 - U1 - U2 - S1 - S2

FOR THREE LEVELS

1.2(D ) LOG A =

( U1 + U2 + U4 - S1 - S2 - S4 ) 0,401 U4 + S4 - U1 - S1

OR //

( D' ) LOG A =

( U2 + U4 + U8 - S2 - S4 - S8 ) 0,401 U8 + S8 - U2 - S2

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 ACTIVITY CANNOT 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, A SATISFACTORY DETERMINATION HAS NOT BEEN OBTAINED .

EXPRESS THE RESULT IN MILLIGRAMS OF ZINC BACITRACIN PER KILOGRAM OF FEEDINGSTUFF .

9 . REPEATABILITY

THE DIFFERENCE BETWEEN THE RESULTS OF TWO DETERMINATIONS CARRIED OUT ON THE SAME SAMPLE BY THE SAME ANALYST SHOULD NOT EXCEED :

- 2 MG/KG, IN ABSOLUTE VALUE, FOR CONTENTS OF ZINC BACITRACIN UP TO 10 MG/KG,

- 20 % RELATED TO THE HIGHEST VALUE FOR CONTENTS FROM 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 .'

( 1 ) 1 MG FEEDINGSTUFF GRADE ZINC BACITRACIN IS EQUIVALENT TO 42 INTERNATIONAL UNITS ( I.U .).

( A ) OTHER METHODS MAY BE USED PROVIDED THAT IT HAS BEEN ESTABLISHED THAT THEY GIVE SIMILAR BACTERIAL SUSPENSIONS .

( B ) ANY COMMERCIAL CULTURE MEDIUM OF SIMILAR COMPOSITION AND GIVING THE SAME RESULTS MAY BE USED .