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Bacterial Endotoxins LAL Test SOP Chromogenic Method
Bacterial Endotoxins LAL Test SOP Chromogenic Method
1. Purpose:-
It is established to provide a procedure for the bacterial endotoxins testing in materials
& products by using endpoint chromogenic method.
2. Scope:-
This procedure is applicable to those materials & products for which endotoxin testing
is required.
3. Responsibilities:-
Quality Control Manager
Microbiologist
Sr. Officer QC
4. Definitions:-
Bacterial Endotoxins: Pyrogens are fever-producing materials that most often
originate from gram-negative bacterial cell walls, but can also originate as leachates
from some chemicals and materials. Pyrogens from bacterial cell walls (the most
commonly encountered type of pyrogen) are referred to as bacterial endotoxin and are
readily detected by Limulus Amebocyte Lysate (LAL) testing systems.
5. Flow Chart:
6. Description / Procedure:
Toxicolor Kit Components:
6.1.1 One vial of endotoxin standard is provided in the kit having potency 0.5EU/ml.
6.1.2 One vial of 2.8ml of LAL Reagent Water is (LRW) also included in the kit for
the reconstitution of standard.
6.1.3 Reconstitute the endotoxin standard with 2.8ml of LRW for the preparation of
0.19EU/ml of endotoxin standard.
6.1.4 Store reconstituted endotoxin solution at 2-8˚C in a refrigerator and don’t use
this solution after 20% decline in absorbance compared with the absorbance on the
first day of reconstitution.
6.3.1 Four vials are provided in the kit by the manufacturer named as 7s, 8s, 9s & 10s.
6.3.2 Transfer whole contents of the vial 7s to vial 8s.
6.3.3 Add 12ml of distilled water in to remaining vials number 9s & 10s.
6.3.4 So we come with three vials named as 8s, 9s & 10s.
6.3.5 Store reconstituted vials at 2-8˚C in a refrigerator
6.4.1 Adjust pH of the sample in between 5.0 – 7.0 with Pyrogen free 0.1 N NaOH or
0.1 N HCl.
6.4.2 In case of dry powder injections, make a 0.1% solution (50mg in 50ml Pyrogen
free water). Fresh water from distillation plant can be used for this purpose.
6.4.3 Mark the tubes as sample, standard & blank.
6.4.4 Dispense 50ul test sample in tube marked as sample, 50ul Pyrogen free water
used during dilution/solution preparation & 50ul of endotoxin standard in tube marked
as standard.
6.4.5 Place the tube stand containing tubes of sample, blank & standard in ice water
bath for 5.0 minutes.
6.4.6 Add 50ul lysate into each tube of sample, blank & standard.
6.4.7 Stir the contents of every tube soon after the addition of lysate for few seconds.
6.4.8 Place the test tube rack in incubator at 30 – 35˚C for 30 – 35 minutes.
6.4.9 After the completion of incubation period place the test tube stand in ice water
bath tray and add 0.25ml of diazo coupling reagents one by one into each test tube
from vial 8s, 9s & 10s. Separate pipette must be used for dispensing of each solution.
6.4.10 Measure absorbance of the test sample and standard against blank at 545±2nm.
8. Record:-
LAL TEST REPORT (End Point Chromogenic Method)
9. References:-
Manufacturer’s Manual