About 70 years ago it was coined that there were two types of Bacteria one Gram – Positive and one Gram-Negative (according to Dr. Hans Christian Gram) both of which can cause several health issues. Gram-negative bacteria freely roam around in our surrounding environment. So it is very much essential to test for the presence of the Endotoxin in medical instruments, including surgical and dialysis water which mainly gets in contact with the human bloodstream. The purpose of this test is to determine endotoxin by gel clot method. This is done using a lysate derived from heamolymph cells of Horse shoe crab Limulus polyphemus . The aim of this work is to find the presence of endotoxin in dialysis water since certain constituent of gram negative bacteria acting as endotoxin may cause serious illness like toxic shock, trauma which is potentially fatal for human.
What is Endotoxin ?
The outer membrane of Gram – negative bacteria with high molecular weight, similar to lipopolysachaarides, very much heat stable and can induce elevated body temperature and illness is Endotoxin. The process of sterilisation doesn’t essentially remove endotoxins since they act as Pyrogens which are usually bacterial products and remains or decaying products of the bacterial cell walls.
Figure: Shows a structure of Gram-negative cell wall where the Lipopolysachharide part containing O-Antigen acts as Endotoxin.
Determination of Endotoxin:
Rabbit Endotoxin Test: Early endotoxin test were being done in rabbit, injecting the samples in rabbits and observing the rise of body temperature, as the rabbit has same endotoxin levels as humans. The process is now outdated as it is time consuming and expensive.
Instead International Pharmacopeia including Indian Pharmacopeia has prescribed several methods which are:Method A: Gel clot Limit Test Method. Method B: Semi-quantitative Gel-Clot Method. Method C: Kinetic turbidimetric Method. Method D: Kinetic Chromogenic Method. Method E: End Point Chromogenic Method.
We have adopted Gel clot Limit Test method as the process is very much accurate, inexpensive and fast. The lysate required for the test is extracted from Horse shoe Crab Limulus Polyphemus found particularly on the beaches of east coast of North America.
Gel Clot Method of Bacterial Endotoxin Determination:
- MATERIALS REQUIRED:
- CONTROL STANDARD ENDOTOXIN
- ENDOTOXIN LYSATE
- WATER BET
- SAMPLE (DIALYSIS WATER)
- WORKING ENDOTOXIN SOLUTION
- HEATING DRY BATH @ 370 C (+/- 1 DEGREE)
The Endotoxin Limit test is expressed by Endotoxin Units (EU/ml). Where the tolerance is upto (2 EU/ml).
We have used the lysate sensitivity upto 0.125 EU/ml
The water used for the test is diluted to 0.125 fold and subsequently being mixed with the lysate with positive and negative controls and positive sample controls. The presence of endotoxin samples results to the clotting as it reacts to the Lysate resulting in coagulation of positive gel clotting.
Tubes 1 & 2 = Negative control, Tubes 3 & 4 Positive endotoxin control, Tubes 5, 6, 8, 9 are test solution, Tubes 7 & 10 positive Sample control, MVD = maximum valid dilution. Samples are denoted as sample X. If 1, 2, 3, 4, 7 or 10 does not show indicated result the test is invalid.
Tubes – 5 & 6 shows no clot but tube 8 & 9 shows clotting as of them one test sample was spiked with known quantities of E. coli (gram negative bacteria) which shows clotting thus the test proves to be very much effective and can be used as method for testing pyrogens in medical industry and dialysis water.
Indian Pharmacopoeia (IP 2010), European Pharmacopoeia, Charles River Laboratories.
Contributed by : Mr. Dipan Roy & Mr. Anukul Das (Biological Division) under Guidance of Dr. Mrinal Kanti Majumdar (Ex. WHO Expert committee member, Geneva).