Scientists use chromogenic, gel clot, and turbidimetric tests to detect bacterial endotoxins in substances such as water, raw materials, equipment, and packaging. Endotoxins are released during cell division or destruction and can cause fever, abnormal clotting, and septic shock. The gel clot test uses Limulus ameboecyte lysate, while the chromogenic test produces a specific color reaction, and the turbidimetric test measures the loss of light intensity. These tests are repeated two or three times for accuracy, and safety parameters vary depending on the substance being tested.
Testing for bacterial endotoxins typically involves the use of reagents which, when mixed into a solution with a possible contaminant, cause a reaction, indicating the presence of an endotoxin. The chromogenic test, gel clot test, and turbidimetric test are the methods commonly employed by scientists for bacterial endotoxin testing. Technicians use these assessment methods on a variety of substances and objects when checking for endotoxin contamination. Water, raw materials used in the manufacture of drugs, equipment and packaging must pass all endotoxin standards.
Bacteria, fungi, and viruses all have protective outer membranes made up of lipopolysaccharides, also called LPS. The lipid portion of these chains contains endotoxins. These substances generally remain within the membrane but are released during the process of cell division and during cell destruction or lysis. In humans, these substances cause fever, abnormal clotting, septic shock, and other symptoms although, unlike exotoxins inside the cell, endotoxins do not convert into a toxoid. Generally, microbiologists check for endotoxins associated with various gram-negative bacteria, including E. coli.
The gel clot test, or Limulus ameboecyte lysate (LAL) assay, involves the use of a membrane-disrupting chemical derived from the amoebocytes of the horseshoe crab, also known as Limulus polyphemus. Technicians observe a positive indication of endotoxins if coagulation or gelling occurs when the lysate is exposed to the object or substance in question. Microbiologists typically use this method of bacterial endotoxin testing in conjunction with chromogenic and turbidimetric testing for conclusive results.
The chromogenic bacterial endotoxin test uses a specially treated LAL. When this lysate comes into contact with an endotoxin, the reaction produces a specific color. The final testing process includes measuring the turbidity or cloudiness of the solution. Technicians expose the solution formed in the gel clot test to a spectrophotometer, which emits a beam of light. By measuring the loss of light intensity in the beam as it passes through the solution, microbiologists can determine whether or not an endotoxin is present.
Lab associates typically complete these three bacterial endotoxin tests two or three times to ensure accurate results. The gel test is not used exclusively as the lower limit of detection is 0.03 Ehrlich units per milliliter (EU/ml). Chromogenic and turbidimetric tests detect endotoxins in the range of 0.005 EU/ml. The safety parameters differ greatly depending on the test substance. While sterile water used for injection or irrigation cannot contain more than 0.25 EU/ml, sterile water for inhalation can contain up to 0.5 EU/ml.
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