Pharmaceutical Division Microbiology

Bacterial Endotoxin (LAL Testing)

Endotoxins are potentially toxic natural compounds of the cell wall of gram negative bacteria. This material is pryrogenic meaning that it can cause high fevers in humans.

The material can also induce acute febrile shock when present in miniscule amounts.

The test for bacterial Endotoxin is used to detect or quantify endotoxins using Limulus Amoebocyte Lysate (LAL) which is an extract of blood cells from the horseshoe crab (Limulus polyphemus).

Two methods for detection of Endotoxin within parenteral preparations and medical devices can be performed at ILS.

Gel Clot Method (Method A) LAL

The gel clot method is based upon the reaction between bacterial endotoxin and a specific lysate. This particular lysate is derived from  the circulating amebocytes associated with the blood-clotting mechanism of the horseshoe crab Limulus polyphemus. (LAL)

If a gel is formed via a clotting reaction, endotoxin is present and the sample fails the test.

Turbidimetric Kinetic Method (Method C).LAL

Turbidity testing determines the cloudiness of a solution by measuring the loss of intensity in a light beam passing through that solution. The cloudiness is caused as the insoluble coagulin concentration increases.

A mixture of the test substance is produced along with the LAL this solution is then illuminated by a spectrophotometer. If a clotting reaction has occurred  the solution will become cloudy. The turbidity is proportional to  the endotoxin concentration.  Specific software is used to measure and analyse the data to determine the onset of turbidity.  A fixed threshold is used and its value decreases with higher endotoxin concentration.

Contact ILS - for more about Bacterial Endotoxin testing and analysis