Microbiological assay of Amino acids.

 

Introduction:

• The microbiological or microbial assay is a type of biological assay in which the relative potency of activity of a compound is determined by measuring the amount required for producing the predicted effect on a suitable test organism under standard conditions. 

• Microbiological assays are time-consuming and also not suitable for all amino acids. 

• Amino acids are essential for the growth and replication of some microorganisms. 

• Many strains of such microbes depend on a particular amino acid. 

• Thus, if such microorganisms are cultured using a small amount of that particular amino acid, a limited degree of growth will be observed, which is measured by turbidimetry or by measuring the increased lactic acid production by either microtitration or pH change. 

• Guthrie and Susi introduced a modified version of microbiological assay utilising diffusion in gels in clinical biochemistry laboratories for screening blood Samples having increased phenylalanine levels. 

• This test was named as the Guthrie test (after its inventors) and is the most widely used microbiological assay method of an amino acid. 

• This bacterial inhibition assay is based on the ability of phenylalanine to counteract the effects of β-2- theienylalanine (a competitive metabolic antagonist on the growth of a special strain of Bacillus subtilis which depends on phenylalanine for its growth.

• The test is carried out on an agar layer in which a mixture of the suspension of B. subtilis spores, minimum amount of growth nutrients, and a fixed amount of β-2 thienylalanine is added. 

• Filter paper discs of 4 mm diameter are soaked in blood and placed on the agar surface along with the blood soaked discs of phenylalanine standards. 

• These agar plates are incubated at 37°C overnight. 

• Bacterial growth is observed only when phenylalanine concentration in the blood discs is sufficient to overcome the effects of β-2 thienylalanine. 

• Growth is observed in zones of growth around each disc. 

• The next day diameter of each zone of growth is measured and related to phenylalanine concentration.