This document describes the Limulus Amoebocyte Lysate (LAL) test used to detect bacterial endotoxins. It involves mixing the LAL reagent, obtained from horseshoe crab blood, with test samples. If endotoxins are present, the LAL reagent will clot, forming a gel. The procedure involves incubating mixtures of the LAL reagent with test samples, positive controls containing endotoxins, and negative controls. After incubation, the mixtures are observed to see if gels formed, indicating the presence of endotoxins above permissible limits. The LAL test is a common method used in the pharmaceutical industry to test for bacterial endotoxins in products.

1. Dr. Imran Sajid

2. Experiment: Bacterial Endotoxin Testing:
Limolus Amoebocytic Lysate Test (LAL Test)
Materials Required
• LAL Reagent Kit (Lysate, standard endotoxin, LRW)
• 5 ml glass tubes, aluminum foil, micropipette, oven, incubator,
test samples

3. Principle (Theoretical Background)
Endotoxins are present in the cell wall of Gram negative
bacteria and are released only on the disintegration of the
cells. They are basically lipopolysaccharides (LPS), present in
the outer membrane of Gram negative bacterial cell wall.
Lipopolysaccharides are made up of three components
1. Lipid A
2. Core Polysaccharide
3. Antigen O
Lipopolysaccharides are not toxic when present in the living
cells, however when cell disintegrate LPS are released from the
cell wall and act as toxins

4. Difference Between Endotoxins and Exotoxins
Endotoxins are the structural part of Gram negative bacterial cell
wall and are released only when cells bursts, while exotoxins are
the toxins which are synthesized in the bacterial cells, and then
living cells release them in to the surrounding medium.
Pharmacological Effects of Endotoxins
There are three major pharmacological effects of endotoxins
1. Pyrogenicity
2. Blood Change
3. Shock

5. 1. Pyrogenicity
It is the ability of a substance to cause change in the body
temperature. The pyrogenic effect of endotoxins is indirect,
they effect the lymphocytes which produce specific
endogenous pyrogenic agents. These agents influence the
hypothalamus part of the brain and increase the body
temperature.
2. Blood Change
When endotoxins are in the body of experimental animals,
they first cause a temporary decrease and then marked
increase in the umber of blood leukocytes.
Endotoxins also damage blood platelets or thrombocytes
which release factors that may cause blood to clot in the
blood vessels, a condition called intravascular clotting or
stroke.

6. More over endotoxins cause increase in the permeability of
the blood capillaries, causing them to leak the fluid portion of
the blood, or some time whole blood, which amy leads to
hemorrhage.
3. Shock
When the Gram negative bacteria are present in large number
in the blood or the endotoxins are injected intravenously
sever shock may occur, with the following symptoms
• Decrease in the blood pressure
• Rapid pulse
• Decreased respiration
• Unconsciousness
High dose may result in circulatory collapse and even death

7. How to test the bacterial endotoxins ?
Endotoxins have been tested in pharmaceutical products by
different methods. The one very traditional method which has
been used in pharma industry or some time is still used for
some specific products, is called Rabbit Pyrogen Test. In this
methods the rabbits are reared in the animal house. The
sample to be tested is injected in the body of the rabbits (test
is performed in triplicate with positive and negative control),
after 1-2 hours change in body temperature is recorded. The
effect of the sample on the body temperature shows the
level/concentration of pyrogens in the sample.
Limolus Amoebocytic Lysate Test
In 1800 Bong and Howel demonstrated that the body fluid of
horse shoe crab (Limolus amoebocyte) clots when it come in
contact Gram Negative bacteria. LAL reagent is prepared from

8. the body fluid of horse shoe crab (Limolus amoebocte,
Trachiplus polyphemous, Trachiplus tridantatus). Currently
followings are the pharmacopeia recommended methods for
bacterial endotoxins testing .
1. Gel Clot Method
2. Chromogenic Method
3. Turbidimetric Method
In this experiment we will perform only gel clot method

9. Procedure

10. Preparation of CSE
Original Stock 0.9ml 0.5ml
0.1ml 0.5ml
5ml
2500 EU
Or
500 EU/ml
50 EU
0.1ml 0.1ml
0.9ml 0.9ml
5 EU 0.5 EU 0.25 EU

11. Gel Clot Method
• Take 5 ml glass tubes, cover them with aluminum foil, and
depyrogenate the tubes (depyrogentaion: 250°C for half hour
or 200 C for 3 hours in the oven).
• Take 0.1 ml lysate in each glass tube
• Add 0.1 ml (100 µl) sample in each of the tube.
• Prepare the relevant positive and negative controls .
• Incubate the tubes at 37°C for 1 hour
Controls
• Positive control = Lysate + CSE
• Negative control = Lysate + LRW
• Positive product control = Lysate + CSE + Product
• Test Sample = Lysate + sample (Three replicates)

12. Results Interpretation
After incubation, gently pick each of the vial and invert it upside
down and observe the gel formation
• Positive control = Gel formation
• Negative control = No gel formation
• Positive product control = Gel formation
• Sample = if there is gel formation = endotoxins above the
permissible limit
• Sample = no gel formation = endotoxins are in the permissible
limit