Bacterial Endotoxins Test LAL Test (End Point Chromogenic Method)

It’s important to note that the Bacterial Endotoxins Test LAL Test (End Point Chromogenic Method), is highly sensitive and specific. It’s crucial to follow proper protocols and standards to ensure accurate and reliable results, especially in industries where product safety is paramount.

The Limulus Amebocyte Lysate (LAL) test is a widely used method for detecting bacterial endotoxins. Endotoxins are heat-stable toxins present in the outer membrane of Gram-negative bacteria. These toxins can cause severe reactions in humans and animals, so it’s crucial to detect and quantify their presence, especially in medical devices and pharmaceutical products.

The LAL test operates on the principle that the LAL enzyme in the horseshoe crab’s blood reacts with bacterial endotoxins, leading to a gel formation. This reaction can be detected visually or measured quantitatively using various methods, including the End Point Chromogenic Method. In this method, the reaction between LAL enzyme and endotoxins produces a color change, and the intensity of the color is proportional to the amount of endotoxin present.


It is established to provide a procedure for the Bacterial Endotoxins Test LAL Test (End Point Chromogenic Method) testing in materials & products by using end point chromogenic method.


This procedure is applicable to those materials & products for which endotoxin testing is required.


  • Quality Control Manager
  • Microbiologist
  • Sr. Officer QC


Bacterial Endotoxins Test LAL Test (End Point Chromogenic Method) 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.
What is LAL Test: Limulus amebocyte lysate (LAL) is an aqueous extract of blood cells (amoebocytes) from the horseshoe crab, Limulus polyphemus. LAL reacts with bacterial endotoxin or lipopolysaccharide (LPS), which is a membrane component of Gram negative bacteria. This reaction is the basis of the LAL test, which is used for the detection and quantification of bacterial endotoxins.

Flow Chart:

  • Reconstitution of endotoxin standard & lysate as per manufacturer’s directions
  • Reconstitution of Diazo coupling reagents as per manufacturer’s directions
  • Dispensing of 50µl each of sample, standard & blank to the tubes marked at sample, standard & blank.
  • Dispensing of 50µl each of sample, standard & blank to the tubes marked at sample, standard & blank.
  • Addition of 0.25ml from vial 8s, 9s & 10s one by one to test tubes marked as sample standard & blank.
  • Measure absorbance at 545±2nm against blank and record results accordingly.


 Toxicolor Kit Components:

Endotoxins Standard

  1. One vial of endotoxin standard is provided in the kit having potency 0.5EU/ml.
  2. One vial of 2.8ml of LAL Reagent Water is (LRW) also included in the kit for the reconstitution of standard.
  3. Reconstitute the endotoxin standard with 2.8ml of LRW for the preparation of 0.19EU/ml of endotoxin standard.
  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 first day of reconstitution.

Toxicolor Lysate (ES 24S or 48S)

  1. Reconstitute the test tube containing lysate with 200µl of buffer provided in the kit. This lysate is sufficient for four tests as 50µl is used per test.
  2. Quickly stir to dissolve. Avoid air bubbling or fuming during stirring.
  3. Be sure that reagent is completely dissolved. This reagent should be reconstituted just before use, however storage of reconstituted lysate must be at -20 to -25˚C in 50ul dispensed quantities in small test tubes. Use stored lysate if color is not changed from white to yellowish.

Diazo Coupling Reagent

  1. Four vials are provided in the kit by the manufacturer named as 7s, 8s, 9s & 10s.
  2. Transfer whole contents of the vial 7s to vial 8s.
  3. Add 12ml of distilled water in to remaining vials number 9s & 10s.
  4. So we come with three vials named as 8s, 9s & 10s.
  5. Store reconstituted vials at 2-8˚C in a refrigerator


  1. Adjust pH of the sample in between 5.0 – 7.0 with Pyrogen free 0.1 N NaOH or 0.1 N HCl.
  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.
  3. Mark the tubes as sample, standard & blank.
  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.
  5. Place the tube stand containing tubes of sample, blank & standard in ice water bath for 5.0 minutes.
  6. Add 50ul lysate into each tube of sample, blank & standard.
  7. Stir the contents of every tube soon after the addition of lysate for few seconds.
  8. Place the test tube rack in incubator at 30 – 35˚C for 30 – 35 minutes.
  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.
  10. Measure absorbance of the test sample and standard against blank at 545±2nm.
 Calculate endotoxins contents in the sample according to following formula:
EU / ml = Potency of standard used × Absorbance of test sample
                               Absorbance of Standard

Testing Frequency:-

Every third batch of each product or as and when required in addition to Bacterial Endotoxins Test LAL Test (End Point Chromogenic Method) by gel clot method.


 LAL TEST REPORT Bacterial Endotoxins Test LAL Test (End Point Chromogenic Method)


Manufacturer’s Manual

Leave a Reply

Your email address will not be published. Required fields are marked *