S.L. 437.82 Art. 6be used. Weigh out and 1,0 ml of about 2. Shake minutes and the pH to 6,5 bromocresol obtain an 6.1.2. Weigh out ml of sodium Shake for 10 centrifuge. 6,5 by means bromocresol volume in a Dilute with 0,42 i.u./ml (= 6.1.3. Weigh out content of 5 sodium phosphate supernatant solution (4.8) indicator.

PREPARATION OF DIE EXTRACT AND ASSAY SOLUTIONS 6.1. Extraction 6.1.1. Premixes and mineral feeds

*Any commercial culture medium of similar composition and giving the same results may a quantity of sample of 2,0 to 5,0 g, add 29,0 ml of the mixture (4.4) sodium sulphide solution (4.9) and shake briefly. Check that the pH is for 10 minutes, add 30 ml of phosphate buffer (4.5), shake for 15 centrifuge. Take a suitable aliquot of the supernatant solution and adjust by means of 1 M hydroxide solution (4.8) with a pH-meter or with the purple solution (4.10) as indicator. Dilute with phosphate buffer (4.5) to expected zinc bacitracin content of 0,42 i.u./ ml (= u8).

Protein concentrates a quantity of sample of 10,0 g, add 49,0 ml of the mixture (4.4) and 1,0 sulphide solution (4.9) and shake briefly. Check that the pH is about 2. minutes. Add 50 ml of phosphate buffer (4.5), shake for 15 minutes and Take a suitable volume of the supernatant solution and adjust the pH to of 1 M sodium hydroxide solution (4.8) with a pH-meter or with the purple solution (4.10) as indicator. Evaporate to approximately half rotary evaporator at a temperature not exceeding 35ºC. phosphate buffer (4.5) to obtain an expected zinc bacitracin content of u ). 8

Other feeds a quantity of sample of 10,0 g (20,0 g for an expected zinc bacitracin mg/kg). Add a mixture of 24,0 ml of the mixture (4.4) and 1,0 ml of sulphide solution (4.9) and homogenize for 10 minutes. Add 25 ml of buffer (4,5), shake for 15 minutes and centrifuge. Take 20 ml of the solution and adjust the pH to 6.5 by means of 1 M sodium hydroxide with a pHmeter or with the bromocresol purple solution (4.10) as to about 4 ml in a rotary evaporator at a temperature not exceeding the residue with phosphate buffer (4.5) to obtain an expected zinc content of 0,42 i.u./ml (= u ). 8 Assay solutions solution u prepare solutions u (expected content: 0,21 i.u./ml), u 8 4 2 content: 0,105 i.u./ml) and u1 (expected content: 0,0525 i.u./ l) by means dilution (1 + 1) with phosphate buffer (4.5).

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

Preparation of the plates through agar is carried out in plates with the four concentrations of the solution (S , S , S and S ) and the four concentrations of the assay 8 4 2 1 u , u and u ). These four concentrations of extract and standard must 4 2 1 placed in each plate. To this effect, select plates big enough to allow holes with a diameter of 10 to 13 mm and not less than 30 mm between made in the agar medium. The test may be carried out on plates a sheet of glass with a faced aluminium or plastic ring placed on top, diameter and 20 mm high. Pour into the plates a quantity of the medium (4.2) inoculated as give a layer about 2 mm thick (60 ml for a plate of 200 mm diameter), a level position, bore the holes and place in them exactly measured volumes and standard solutions (between 0,10 and 0,15 ml per hole, according diameter). Apply each concentration at least four times so that each determination to an evaluation of 32 zones of inhibition. 7.3. Incubation

Incubate the plates for 16 to 18 hours at 30 ± 2ºC.