Appendix (to Annex II)

See 6.1.

Proceed as specified in 6.2. but using the fat-extraction tubes (see 5.6.).

The test portion shall be delivered as completely as possible at the bottom of the extraction tube.

See 6.3.

See 6.4.

Add 2 ml of the ammonia solution (4.1.), or an equivalent volume of a more concentrated ammonia solution, and mix thoroughly with the pretreated test portion at the bottom of the tube. After the addition of the ammonia, carry out the determination without delay.

Add 10 ml of the ethanol (4.2.) and mix gently but thoroughly at the bottom of the tube. If desired, add two drops of the Congo red or Cresol red solution (4.3.).

Add 25 ml of diethyl ether (4.4.), close the tube with a cork saturated with water or with a stopper wetted with water (5.6.), and shake the tube vigorously, but not excessively (in order to avoid the formation of persistent emulsions), with repeated inversions for one minute. If necessary, cool the tube in running water, then carefully remove the cork or stopper and rinse it and the neck of the tube with a little of the mixed solvent (4.6.) using the wash bottle (5.8.) so that the rinsings run into the tube.

Add 25 ml of light petroleum (4.5.), close the tube with the rewetted cork or stopper (rewet by dipping in water), and shake the tube gently for 30 seconds as described in A.1.5.3.

Centrifuge the closed tube for one to five minutes at a rotational frequency of 500 to 600 rev min^—1 (5.2.). If the centrifuge is not available (see note to 5.2.), allow the closed tube to stand in the rack (5.7.) for at least 30 minutes until the supernatant layer is clear and distincly separated from the aqueous layer. If necessary, cool the tube in running water.

Carefully remove the cork or stopper and rinse it and the neck of the tube with a little of the mixed solvent so that the rinsings run into the tube.

Carefully transfer the supernatant layer out of the tube into the prepared fat-collecting vessel (6.4.) containing a few boiling aids (5.10.) in the case of flasks (optional with metal dishes), avoiding the transfer of any of the aqueous layer. Rinse the outlet of the fitting with a little of the mixed solvent, collecting the rinsings in the fat-collecting vessel.

Rinse the outlet of the fitting with a little of the mixed solvent, collecting the rinsings in the vessel. If desired, the solvent or part of the solvent may be removed from the vessel by distillation or evaporation as described in 6.5.12.

Again loosen the fitting from the neck, slightly raise the fitting and add 5 ml of the ethanol to the contents of the tube, using the ethanol to rinse the long inner limb of the fitting; mix as described in A. 1.5.2.

Carry out the second extraction by repeating the operations described in A.1.5.3. to A.1.5.8., but using only 15 ml of diethyl ether (4.4.) and 15 ml of light petroleum (4.5.). Use the ether to rinse the long inner limb of the fitting during the removal of the fitting from the tube after the previous extraction.

The third extraction can be omitted for skimmed milk.

Proceed as described in 6.5.12. to 6.5.16.