S.L. 437.82 ст. 4§ emulsion. choride a.p. ml of water. suspensions. may be used, e.g. may be used, e.g. 4.6. 4.7. 4.8. potassium methanol (4.4). 4.9. 41600) or 4.10. 41010) or 4.11. Glass 4.12. pH 4.13. 4.14. 5. 5.1. Glass 200 mm, fitted glass joint (to 5.2. 5.3. 250 5.4. 6. Dissolve an methanol stable for up with 5

CULTURE MEDIA AND REAGENTS † 4.1. Culture medium Meat peptone 6ꞏ0 g

Tryptone 4ꞏ0 g Yeast extract 3ꞏ0 g Meat extract 1ꞏ5 g

Glucose 1ꞏ0 g Agar 15ꞏ0 g Water 1 000 ml pH 6ꞏ5 (after sterilization) 4.2. Assay medium 4.2.1. ‡ Base layer Meat peptone 6ꞏ0 g Yeast extract 3ꞏ0 g Meat extract 1ꞏ5 g

Agar 10ꞏ0 g Water 1 000 ml pH 6ꞏ5 (after sterilization)

4.2.2. Seed layer

As for point 4.1, with the addition of 2ꞏ0 g of silicone anti-foaming 4.3. Sodium chloride solution 0ꞏ4 % (w/v): dissolve 4 g of sodium in water and dilute to 1 000 ml; sterilize. 4.4. Methanol, pure.

4.5. Methanol 50 % (v/v): dilute 500 ml of methanol (4.4) with 500

*Other methods may be used provided it has been established that they give similar bacterial †Any commercial culture medium of similar composition and giving the same results oxoid antibiotic medium 1 (CM 327) with an addition of oxoid agar No 3 (L 13). ‡Any commercial culture medium of similar composition and giving the same results oxoid antibiotic medium 2 (CM 335) with an addition of oxoid agar No 3 (L 13). §e.g. SE 2 from Wacker Chemie GmbH, Munich. Methanol 80% (v/v): dilute 800 ml of methanol (4.4) with 200 ml of water.

Tris(hydroxymethyl) aminomethane a.p. Potassium chloride methanolic solution 1ꞏ5% (w/v): dissolve 1ꞏ5 g of chloride a.p. in 20 ml of water, make up the volume to m 100 ml with

Cation exchanger: Dowex 50 W×8, 20 to 50 mesh, Na form (cat. Serva No equivalent.

Anion exchanger: Dowex 1×2, 50 to 100 mesh, Cl form (cat. Serva No equivalent. Before use, keep for 12 to 14 hours in 80% methanol (4.6).

wool. indicator paper (pH 6ꞏ6 to 8ꞏ1). Ascorbic acid.

Standard substance: flavophospholipol of known activity. APPARATUS tube for chromatography, internal diameter: 9 mm, length: 150 to with a stopcock at the narrowed part of the lower end and a ground-connect with the dropping funnel (5.2)) at the upper end. Dropping funnel 250 ml, fitted with a stopcock and a ground-glass joint. ml conical flask with ground-glass joint.

Reflux condenser with ground-glass joint. STANDARD SOLUTIONS accurately weighed quantity of the standard substance (4.14) in 50% (4.5) and dilute to give a stock solution containing 100 µg flavophospholipol per millilitre. Stored in stoppered flasks at 4ºC this solution is to two months. From this stock solution prepare by successive dilution methanol (4.5) the following solutions: µg/ml

µg/ml

µg/ml

µg/ml

PREPARATION OF THE EXTRACT Extraction

Concentrates, premixes and mineral feeds a quantity of sample of 2ꞏ0 to 5ꞏ0 g and add about 150 mg of ascorbic Homogenize with 150 ml of 50% methanol (4.5) in a conical flask (5.3) the pH to 8ꞏ1 to 8ꞏ2 with about 400 mg of tris(hydroxymethyl) (4.7). Check the pH with indicator paper (4.12). Allow to stand for 15 readjust the pH to 8ꞏ1 to 8ꞏ2 with tris(hydroxymethyl) aminomethane for 10 minutes under reflux (5.4) with constant stirring. Allow to cool, mixture and decant the extract.

Other feeds a quantity of sample of 5ꞏ0 to 30ꞏ0 g containing at least 30 µg of flavophospholipol. Homegenize with 150 ml of 50% methanol (4.5) flask (5.3) and adjust the pH to 8ꞏ1 to 8ꞏ2 with about 400 mg of tris(hydroxymethyl) aminomethane (4.7). Check the pH with indicator paper (4.12). Allow minutes, then readjust the pH to 8ꞏ1 to 8ꞏ2 with tris(hydroxymethyl)aminomethane (4.7) and boil for 10 minutes under reflux (5.4) with constant stirring. centrifuge the mixture and decant the extract. 7.2. Purification (this step may be omitted for concentrates, mineral feeds) Mix 110 ml of the extract with 11 g of the cation exchanger (4.9), minute under reflux (5.4) with constant stirring. Separate the cation centrifugation or filtration. Mix 100 ml of the extract with 150 ml of and store the solution for 12 to 15 hours at 4ºC. Filter off the flocculent cold. Insert a glass wool plug (4.11) at the bottom end of a glass tube (5.1), tube 5 ml of the anion exchanger (4.10) and wash the column with methanol (4.6). Using the funnel (5.2), transfer to the column a volume at least 100 ml which is expected to contain 16 µg of flavophospholipol a 30 g sample of feedingstuff at 1 ppm). Where necessary, before application column, dilute the filtrate with 80% methanol (4.6) to obtain flavophospholipol content of 16 µg/100 ml. Adjust the flow rate minute. Discard the effluent. Then wash the column with 50 ml of (4.6) and discard the effluent. Elute the flavophospholipol with the solution of potassium chloride (4.8) keeping the flow rate to about Collect 50 ml of the eluate in a graduated flask, add 30 ml of water solution should have a flavophospholipol content of 0ꞏ2 µg/ml (= U ). 8 7.3. Assay solutions Where necessary (i.e. when the purification step has been omitted), extract obtained in point 7.1.1 with 50% methanol (4.5) to obtain flavophospholipol content of 0ꞏ2 µg/ml (= U ). From solution U prepare 8 8 U (expected content: 0ꞏ1 µg/ml), U (expected content: 0ꞏ05 µg/ml) 4 2 (expected content: 0ꞏ025 µg/ml) by means of successive dilution (1 methanol (4.5).