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Methods for Standardization of Amino Acids (Microbiological Assay)

  • 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:

Standardization of Amino Acids
Standardization of Amino Acids

1) Agar Diffusion Assay (Standardization of Amino Acids)

Principle:

  • Based on the diffusion of amino acids through an agar medium and their effect on the growth of a specific microorganism.

Procedure:

a) Preparation:

  • Inoculate an agar plate with a standardized suspension of an amino acid-dependent microorganism.

b) Application:

  • Place wells or paper discs containing the amino acid samples on the agar surface.

c) Incubation:

  • Incubate the plates to allow bacterial growth and amino acid diffusion.

d) Measurement:

  • Measure the diameter of growth zones around the wells or discs.

e) 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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