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Standardization of Amino Acids using microbiological assays involves measuring the response of specific microorganisms that require particular amino acids for growth.
Here's an overview of the methods typically used for this purpose:
1) Agar Diffusion Assay
Principle
Based on the diffusion of amino acids through an agar medium and their effect on the growth of a specific microorganism.
Procedure
1) Preparation
Inoculate an agar plate with a standardized suspension of an amino acid-dependent microorganism.
2) Application
Place wells or paper discs containing the amino acid samples on the agar surface.
3) Incubation
Incubate the plates to allow bacterial growth and amino acid diffusion.
4) Measurement
Measure the diameter of growth zones around the wells or discs.
5) Comparison
Compare the growth zones with those produced by standard amino acid solutions to determine concentration.
Applications
Commonly used for determining the concentration of essential amino acids in samples.
2) Turbidimetric Assay
Principle
Measures the growth of a microorganism in a liquid culture medium by assessing the turbidity (cloudiness) of the medium, which correlates with the concentration of the amino acid.
Procedure
1) Preparation
Prepare a series of test tubes with a liquid growth medium containing varying known concentrations of the amino acid to create a standard curve.
Inoculate each tube with a standardized amount of an amino acid-dependent microorganism.
2) Sample Testing
Prepare additional test tubes with the same growth medium and inoculum, adding the unknown amino acid samples.
3) Incubation
Incubate all tubes under controlled conditions (temperature, time) to allow microbial growth.
4) Measurement
Measure the turbidity of each tube using a spectrophotometer at a specific wavelength.
5) Comparison
Compare the turbidity of the test samples with the standard curve to determine the concentration of the amino acid.
Applications
Suitable for determining concentrations of amino acids such as lysine, methionine, and tryptophan.
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