9. Pre-test preparations

#### 9.1 pH of the sample

Example: If the product endotoxin limit is 1 EU/ml, then we have to make the

Since we are using 0.25 EU/ml, this is called lambda. Divide the endotoxin limit

One test tube 1:3 The result should be positive

Third 1:5 The result should be negative

Second 1:4 The result may be positive or negative

dilution as follows:

of product with lambda 1/0.25 = 1:4

As per USP, we have to test 3 test as follows:

This means the product is passed.

Growing and Handling of Bacterial Cultures

Chromogenic lysate [2], Respective endotoxin standard,

8.5.7 Preparation of acetic acid 0.8 m

storage.

102

Toxicolor lysate

8.5.8 Diazo coupling reagent

8.5.6 LAL test reagents (chromogenic method)

Diazo coupling reagent (set of four bottles).

During stirring solution must not touch the foil.

Note: Stir every time vigorously before use.

vial in ice water bath for 2–3 min before use.

Reconstituted lysate may only be deep frozen once.

Note: All reagents must be stored in refrigerator at 2–8°C.

Dissolve 45.6 ml of acetic acid in 1 liter of distilled water. The final concentration of acetic acid is 0.8 M. This solution can be stored for 3 months. Remove the plastic cover. Wipe off with 70% alcohol around the rubber cap and top portion of every vial. Remove the aluminum cap with sterile and pyrogen free forceps and then cover with depyrogenated aluminum foil to avoid any Endotoxin contamination. (2.8 ml LAL water vial is provided with Endotoxin vial, concentration is mentioned on the label). Pour whole quantity of LAL water into the ET vial and cover with foil. Mix vigorously for at least 10 s by vortexer.

Storage: Store reconstituted Endotoxins solution at 4°C in a refrigerator for 14 days. The solution can also be stored at –20°C for a month. Avoid freezing during

(Buffer vial 0.35 ml and LAL water are provided with Lysate. Sensitivity is mentioned on the certificate). After taking from the refrigerator, pour whole quantity of buffer and 0.35 ml LAL water into the lysate vial as soon as possible, covers with foil. Then quickly stir to dissolve. Avoid air bubbling during stirring. Place the

Note: Be sure that the reagent is completely dissolved. This reagent must be reconstituted just before use. The reagent is extremely sensitive and must be consumed at one time. Storage should be avoided, but can be stored at 20°C in 0.1 ml dispensed quantities in small test tubes. Use stored lysate if the color is not changed.

Four bottles are provided with one set, marked as 7, 8, 9 and 10s respectively. Transfer whole quantity of bottle no. 7 s into bottle no. 8 s. Then add 12 ml distilled

The pH of the sample is adjusted by pyrogen free 0.1 N NaOH or 0.1 N HCl. The pH of the sample should be between 6.0 and 8.0.

#### 9.2 Test tubes settings

Arrange test tubes in two stands as under; stand 1—test tubes for sample and standard dilutions; stand 2—test tubes for reaction.
