Water BET Test (Purified and WFI)

Requirements 

Ø  Take the Depyrogenate glassware (250deg C temperature at least 1 hour).

Ø  Be ensure all material are Pyrogen free.

Ø 10x75 mm depyrogenated incubation glass tubes

Ø 16x100 mm depyrogenated dilution glass tubes

Ø 0.125 EU/ml Bacterial Endotoxin test kit from Charles River

Ø 20-200µl depyrogenated micropipette tips

Ø 100-1000µl depyrogenated micropipette tips

Ø Dry heat block calibrated at 37±1⁰C temperature

Method :1

Ø  Water Endotoxin Release Limit (ERL) = 0.25 EU/ml

Ø  Water potency (C)=1 mg/1ml.

Ø  (LAL ) Lysate Sensitivity (λ)= 0.125 EU/ml

Ø  Select the MVD (Maximum Valid Dilution) method.

Ø  Calculate as below

Test Sample:

MVD = ERL X C/ λ

= 0.25 x 1/0.125

=2 (dilution factor)

Ø  Perform 2 dilution of water.(it means 1 dilution water sample and 1 dilution LRW).

Ø  Next take the 50µl of sample, 50µl of LAL reagent water , and 100µl of Lysate (LAL).

Ø  Incubate at 37±1⁰C  at 1 hour .

Ø  Observe the reading.

ERL Verification :

= Lambda X Dilution factor = ERL

=0.125 X 2 = 0.25

Negative Control:

Ø  Next take the 100 µl of LRW and 100µl of Lysate (LAL).

Ø  Incubate at 37±1⁰C 1 hour .

Ø  Observe the reading.

Positive Control :

Ø  Take the 100 µl (ERL X 2) CSE  and 100µl of Lysate (LAL).

Ø  Incubate at 37±1⁰C  at 1 hour .

Ø  Observe the reading

Conclusion: 

After incubation read the test tubes by gently inverting them at 180⁰. The presence of a gel clot that remains in the bottom of the test tube after inversion indicates a positive test. The test is negative if the clot breaks apart or if the contents of the tube remain liquid

Method :2

Ø  Water Endotoxin Release Limit (ERL) = 0.125 EU/ml

Ø  Water potency (C)=1 mg/1ml.

Ø  (LAL ) Lysate Sensitivity (λ)= 0.125 EU/ml

Ø  Select the MVD (Maximum Valid Dilution) method.

Ø  Calculate as below

Test Sample:

 MVD = ERL X C/ λ

= 0.125 x 1/0.125

= 1(dilution factor)

Ø  Perform 1 dilution of water. (not required dilution)

Ø  Ø  Next take the 50µl of sample, 50µl of LAL reagent water , and 100µl of Lysate (LAL).

Ø  Incubate at 37±1⁰C  at 1 hour .

Ø  Observe the reading.

ERL Verification :

= Lambda X Dilution factor = ERL

=0.125 X 1 = 0.125

Negative Control:

Ø  Next take the 100 µl of LRW and 100µl of Lysate (LAL).

Ø  Incubate at 37±1⁰C  at 1 hour .

Ø  Observe the reading.

Positive Control :

Ø  take the 100 µl (ERL X 2) CSE and 100µl of Lysate (LAL).

Ø  Incubate at 37±1⁰C  at 1 hour .

Ø  Observe the reading

Conclusion: 

After incubation read the test tubes by gently inverting them at 180⁰. The presence of a gel clot that remains in the bottom of the test tube after inversion indicates a positive test. The test is negative if the clot breaks apart or if the contents of the tube remain liquid

Method :3

Ø  Water Endotoxin Release Limit (ERL) = 0.125 EU/ml

Ø  Water potency (C)=1 mg/1ml.

Ø  (LAL ) Lysate Sensitivity (λ)= 0.03125 EU/ml

Ø  Select the MVD (Maximum Valid Dilution) method.

Ø  Calculate as below

Test Sample:

 MVD = ERL X C/ λ

=0.125 x 1/0.03125

= 4 (dilution factor)

Ø  Perform 4 dilution of water.(it means 1 dilution water sample and 3 dilution LRW).

Ø  Ø  Next take the 50µl of sample, 50µl of LAL reagent water , and 100µl of Lysate (LAL).

Ø  Incubate at 37±1⁰C  at 1 hour .

Ø  Observe the reading.

ERL Verification :

= Lambda X Dilution factor = ERL

=0.03125 X 4 = 0.125

Negative Control:

Ø  Next take the 100 µl of LRW and 100µl of Lysate (LAL).

Ø  Incubate at 37±1⁰C  at 1 hour .

Ø  Observe the reading.

Positive Control :

Ø  take the 100 µl (ERL X 2) CSE and 100µl of Lysate (LAL).

Ø  Incubate at 37±1⁰C  at 1 hour .

Ø  Observe the reading

Conclusion: 

After incubation read the test tubes by gently inverting them at 180⁰. The presence of a gel clot that remains in the bottom of the test tube after inversion indicates a positive test. The test is negative if the clot breaks apart or if the contents of the tube remain liquid