10. Procedure

#### 10.1 Sample preparations

Take 0.05 ml well-mixed sample into small test tubes. If required, make 1/10 dilution of the sample with Pyrogen free water as Below, Take 4.5 ml of pyrogen free water in the test tube. Then add 0.5 ml of well-mixed sample. Vortex mixing for a few seconds.

Take 0.1 ml into a small test tube for further process.

#### 10.2 Standard preparation

Make a dilution of the endotoxin (concentration 0.470 EU/ml) according to the product limit. For making 0.235 EU/ml (if the product limit is 0.25) proceed as follows;

Take 0.05 ml of the reconstituted endotoxin in the test tube after stirring. Add 0.05 ml of pyrogen free water and vortex to mix. Now the final dilution is 0.235 EU/ml.

Take 0.05 ml of step 2 into a small test tube for further process.

#### 10.3 Blank preparation

Pour 0.05 ml of pyrogen free water (being used in the test) in small test tube as a blank for further process.

Lysate addition

Place the tube stand for small test tubes (containing the tubes of blank, standard and diluted samples) in ice water bath or suitable ice water container. Add 0.05 ml of lysate to all of the tubes as soon as possible. Stir the contents of every tube soon after the addition of lysate for a few seconds. Avoid foaming.

#### 10.4 Incubation

Soon after the addition of lysate, place the test tube rack in the incubator set at 32.5 + 2.5°C for 30 min. The tube rack can be placed in the water container placed in the incubator.
