Enterobacteriaceae are mostly gram-negative bacilli with endotoxin. If they cause sepsis, meningitis and other infections, endotoxin can be detected in blood, cerebrospinal fluid and body fluids to assist in clinical diagnosis. In addition, in the production of pharmaceuticals and other processes, endotoxin-producing bacteria in the natural environment often contaminate injectable preparations, biological products, etc., and should be strictly inspected before leaving the factory. The horseshoe crab test can measure trace amounts of endotoxin (0.1-1.0 ng/ml).

Operation method

horseshoe crab test

Principle

Horseshoe crab is a kind of marine arthropod, the blood contains a kind of deformed cell, the lysate of this cell can react with trace bacterial endotoxin to form a gel, that is, an enzyme in the cell lysate is activated by the endotoxin, so as to make the proteins in the cell lysate form a gel. The horseshoe crab test has the advantages of being rapid, simple and sensitive.

Materials and Instruments

Horseshoe crab reagent
Saline Sterile water Endotoxin
Ampoules Habits Water bath

Move

I. Materials
1. Horseshoe crab reagent (i.e., lyophilized product of horseshoe crab transformed cell lysate in ampoules).
2. Substance to be measured (blood, bacterial culture supernatant or injection, etc.).
3. Standard endotoxin (E. coli endotoxin content 100 ng/ml), pyrogen-free saline, sterile distilled water.
4. 1 ml sterile pipette, 37°C water bath, etc.
II. Methodology
1. Open 3 ampoules of horseshoe crab reagent, add 0.1 ml of sterile distilled water to each to dissolve them, and number them ①, ② and ③ at the same time.
2. Add 0.1 ml each of the substance to be measured, standard endotoxin and pyrogen-free saline to ①, ② and ③ ampoules, respectively.
3. After gently shaking, put it vertically in 37℃ water bath, and take it out after 15-30 min to observe whether there is any solidification.
III. Observation of results
X X Form a firm gel and hold the ampoule gel upside down without moving it.
X Forms a gel, but not firm, holding the ampoule upside down the gel can move.
- No gel formation.
Any positive result indicates endotoxin positivity, ② for positive control and ③ for negative control in this experiment.

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