Bacterial Endotoxin Test by Gel-Clot Method

Copyright © 2019 by author(s) and International Journal of Trend in Scientific Research and Development Journal. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0) (http://creativecommons.org/licenses/ by/4.0) ABSTRACT The Bacterial Endotoxin Test (BET) is a test to quantify Endotoxin from ‘Gramnegative bacteria using ‘amoebocyte lysate’ extracted from ‘Limulus polyphemus or Tachypleus tridentatus’ i.e. horseshoe crab. Three techniques are used for the confirmation of endotoxins, those are: the gel-clot technique, the turbidimetric technique, and the chromogenic technique. Endotoxins are toxic complexes which is invariably associated with the cell-wall of the Gram-negative bacteria irrespective of bacterial pathogenicity. Endotoxins are rarely fatal, but they cause fever and hence Endotoxin carrying bacteria are known as ‘Pyrogen’.


INTRODUCTION
Endotoxins are toxic complexes which is invariably associated with the cell-wall of the Gram-negative bacteria irrespective of bacterial pathogenicity. Endotoxins are rarely fatal, but they cause fever and hence Endotoxin carrying bacteria are known as 'Pyrogen'. Endotoxin is toxic substances which is bound with the bacterial cell wall and are released when the bacterium disintegrates. Endotoxin composed of lipopolysaccharide and lipoprotein complexes. The components that are protein determine its antigenic property; and the polysaccharide component determines the antibody type that can react with the Endotoxin molecule. The reaction of polysaccharide components with the Endotoxin when allowed produces an immune reaction. Endotoxin is rarely fatal, although they often cause fever.
Thus, Bacterial Endotoxin test is the confirmatory test that assures the presence or absence of the Endotoxin in the medicine sample. Mostly bacterial Endotoxin test are performed for the inject-able pharmaceutical products, e.g. SWFI (Sterile Water for Injection), Normal Saline (Sodium Chloride Sterile) and other injections like Amikacin sulphate, Ascorbic acid, Esmoprazole sodium, Methylprednisolone, Iron sucrose, Ondansetron, Ranitidine hydrochloride, Thiopental sodium sterile, etc. All these products have their specific Endotoxin limit value determined by 'EU/mg'.

GRAM-NEGATIVE BACTERIA
In the method of bacterial differentiation such bacteria do not retain the crystal violet stain used in the gram-staining. These bacteria are characterized by their cell envelopes. Their cell wall are composed of a thin peptidoglycan cell-wall sandwiched between an inner cytoplasmic cell membrane and a bacterial outer membrane.
Gram-negative bacteria are found almost everywhere. The gram-negative bacteria include the model organism Escherichia coli, as well as many pathogenic bacteria. Their outer membrane protects them from many antibiotics (including penicillin); detergents that would normally damage the peptidoglycans of the (inner) cell membrane; and lysozyme, an antimicrobial enzyme produced by animals that forms part of the innate immune system. Additionally, the outer leaflet of this membrane comprises a complex lipopolysaccharide (LPS) whose 'lipid A' component can cause a toxic reaction when these bacteria are lysed by immune cells. This toxic reaction can include fever, an increased respiratory rate, and low blood pressure -a lifethreatening condition known as septic shock.
Some examples of Gram-negative bacteria that have demonstrated drug resistance include  E. coli, which causes the majority of urinary tract infections  Acinetobacter baumanii, which causes disease mainly in healthcare settings. In addition, wound infections caused by Acinetobacter have been found in U.S. military personnel who were deployed to Iraq and Afghanistan.
 Pseudomonas aeruginosa, which causes bloodstream infections and pneumonia in hospitalized patients. It is a common cause of pneumonia in patients with cystic fibrosis.  Klebsiella pneumoniae, which causes many types of healthcare-associated infections, including pneumonia, urinary tract infections, and bloodstream infections  Neisseria gonorrhoeae, which causes the sexually transmitted disease gonorrhea, is the second most commonly reported infectious disease in the United States.

ENDOTOXINS
The bacteria those are associated with Endotoxin grows side by side with fungus and or mold in watery environments specifically. These gram negative micro-organisms (bacteria) collectively get accumulated in the human digestive system and thus these microorganisms are responsible for the inflammation in the gut and also in different parts of the body. Endotoxin had been authenticated as inflammatory agent, onset clinical diabetes, obesity, nausea, vomiting, diarrhea, fever, disseminated intravascular coagulation, vascular collapse, and organ failure.
Endotoxins are thus very necessary to get removed eliminated out from the inject-able pharmaceutical products.    Table-02, all the respective reaction tubes are incubated for 60minutes, at 37 º C, in the heating mantle. 2. After the completion of 60minutes, the incubation of the reaction tubes are over, and thus it is necessary to remove the reaction tubes one by one carefully and is inverted about 180º. 3. The tubes are then to be observed and the result of the reaction in each of the respective tubes has to be recorded. As if the clot formation takes place in the reaction tubes, then it means Endotoxin was present in that reaction tube and thus the result is considered to be positive. And if the clot formation doesn't takes place, then it means Endotoxin was absent in that reaction tube and thus the result is considered to be negative.

MAXIMUM VALID DILLUTION (MVD)
The maximum allowable dilution of a sample at which the Endotoxin Limit can be determined is known as the maximum valid dilution.

ENDOTOXIN LIMIT
The endotoxin limit of parenteral drug, defined on the basis of dose, equals K/M 2 , where K is a threshold pyrogenic dose of endotoxin per kg of body weight. When the product is injected at frequent intervals or infused continuously, M is the maximum total dose administered in a single hour period. The endotoxin limit of the parenteral drugs is specified in the individual monograph in units such as EU/ml, EU/mg, EU/Unit of biological activity, etc.
Parenteral drugs are the sterile preparations containing one or more active ingredients intended to be administered to a patient other than the alimentary canal (gastrointestinal tract) routes.
The concentration of the sample solution: 1. mg/ml: in the case of endotoxin limit particularized by weight (EU/mg); 2. ml/ml: in the case of endotoxin limit particularized by volume (EU/ml); 3. Units/ml: in the case of endotoxin limit particularized by unit of biological activity (EU/unit); 4. λ: the labeled sensitivity in the Gel-Clot technique (EU/ml). Following are the few parenteral drugs raw material and their endotoxin limits:   All the respective reaction tubes are incubated for 60minutes, at 37 º C, in the heating mantle. 2. After the completion of 60minutes, the incubation of the reaction tubes are over, and thus it is necessary to remove the reaction tubes one by one carefully and is inverted about 180º. 3. The tubes are then to be observed and the result of the reaction in each of the respective tubes has to be recorded. As if the clot formation takes place in the reaction tubes, then it means Endotoxin was present in that reaction tube and thus the result is considered to be positive. And if the clot formation doesn't takes place, then it means Endotoxin was absent in that reaction tube and thus the result is considered to be negative.