Olive Oil Substrate Emulsion

Homogenise 40 mL of olive oil, 330 mL of acacia solution and 30 mL of water in an 800-mL beaker placed in a vessel containing a mixture of ice and ethanol as cooling mixture. Emulsify using a mixer at an average speed of 1000 to 2000 revolutions per minute. Cool to 5° to 10°. Increase the mixing speed to 8000 revolutions per minute. Mix for 30 minutes keeping the temperature below 25° by the continuous addition of crushed ice into the cooling mixture (a mixture of calcium chloride and crushed ice is also suitable). Store this preparation (the stock emulsion) in a refrigerator and use within 14 days. The emulsion must not separate into two distinct layers. Check the diameter of the globules of the emulsion under a microscope. At least 90% have a diameter below 3 µm and none has a diameter greater than 10 µm. Shake the emulsion thoroughly before preparing the substrate emulsion.

For 10 determinations mix the following solutions in the order indicated: 100 mL of the stock emulsion, 80 mL of tris-chloride buffer solution, 20 mL of a freshly prepared 8% w/v solution of sodium taurocholate EPBRP and 95 mL of water.

Use on the day of preparation.