Bacterial Endotoxin Testing Explained
How the LAL assay quantifies endotoxin burden, what EU per milligram means, and why the test needs its own vial.
What is bacterial endotoxin testing?
Endotoxin testing quantifies lipopolysaccharide from the outer membrane of Gram-negative bacteria. The kinetic chromogenic LAL method uses an enzyme cascade derived from horseshoe crab amoebocyte lysate, which endotoxin activates, releasing a coloured marker. The rate of colour development is proportional to endotoxin concentration, reported in endotoxin units per milligram.
Endotoxin is not a living organism, so it survives sterilisation. A sample can be perfectly sterile and still carry a high endotoxin burden, which is why the two assays are not substitutes for one another.
Why the positive product control matters
Peptide samples can interfere with the LAL cascade, either inhibiting it and producing a falsely low result, or enhancing it and producing a falsely high one.
A valid endotoxin test therefore includes a positive product control: a known quantity of endotoxin spiked into the sample matrix. If the spike recovers within the accepted range, the matrix is not interfering and the sample result is trustworthy.
A certificate reporting an endotoxin figure without a positive product control has not demonstrated that the number means anything for that particular material.
Frequently asked questions
- What is the difference between sterility and endotoxin testing?
- Sterility testing asks whether viable organisms are present and capable of growing. Endotoxin testing quantifies a heat-stable bacterial cell-wall fragment that remains after the organisms are dead. Because endotoxin survives sterilisation, material can pass sterility and fail endotoxin. The two assays answer genuinely different questions and neither substitutes for the other.
- What does EU/mg mean on a certificate?
- Endotoxin units per milligram of peptide. An endotoxin unit is a standardised measure of biological activity rather than of mass, calibrated against an international reference standard, because endotoxin potency varies with molecular structure. Expressing the result per milligram of peptide rather than per millilitre of solution makes results comparable across different reconstitution volumes.
About the author
REPLACE: Microbiology Lead
Head of Microbiology · MSc Microbiology
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