With a sterile pipette,  aseptically add 0.5ml of 0.067M phosphate buffer, pH 7.0 to 16 x 125 mm screw-cap tubes

Suspend several loopfulls of test cultures in the buffer solution, using sterile loops

Place the tubes containing the emulsified cultures in a previously heated water bath at 68EC for 20 minutes. Time and temperature are critical

Remove the tubes from heat and allow to cool to room temperature

Add 0.5ml of the freshly-prepared Tween-peroxide mixture to each tube and replace caps loosely

Observe the formation of bubbles appearing on the surface of the liquid. Do not shake the tubes because Tween 80 also may form bubbles when shaken, resulting in false positive results

Hold negative tubes for 20 minutes before discarding