Lal test for endotoxin procedure

LAL test or Limulus Amebocyte Lysate is widely used to detect the presence of bacterial endotoxins. Endotoxins are found in the cell walls of gram-negative bacteria.

Method and Materials for lal test in microbiology

• LAL Reagent: Limulus Amebocyte Lysate reagent,
• Control Standard Endotoxin (CSE): A known concentration of endotoxin used as a positive control.
• Sample: Sample to be tested.
• Test Tubes or Microplates: To conduct the reactions.
• Water Bath or Incubator: To control the temperature during the test.
• Spectrophotometer or other measuring instrument: To measure the reaction.

Lal test procedure

Preparation

1. Pre-warm the LAL reagent: If required, warm the LAL reagent to the specified temperature (usually 37°C) using a water bath.
2. Prepare the Sample:
   • If the sample is in solid form, dissolve it in a suitable solvent.
   • Ensure the sample is within the specified pH range for the LAL reagent.

Controls

1. Positive Control:
   • Prepare a series of tubes or wells with known concentrations of Control Standard Endotoxin (CSE). This serves as a positive control to validate the sensitivity of the LAL reagent.
2. Negative Control:
   • Include a tube or well with LAL reagent and no endotoxin as a negative control.

Reaction Setup

1. Add LAL Reagen: Dispense the LAL reagent into tubes or wells. Use a separate tube or well for each sample and control.
2. Add Sample: Add the sample to the appropriate tubes or wells. Mix gently.
3. Incubation: Incubate the reaction mixture at the specified temperature (usually 37°C) for a defined period (commonly 1 hour).

Lal test Result

After incubation, observe for gel formation, color change, or other indications of a positive reaction.

NOTE: Measure the reaction using a spectrophotometer. The degree of reaction is proportional to the concentration of endotoxin.