Microbiological assays were used for quantitation of amino acids since many years as they were the most reliable, and specific available tests. They can be used for any type of biological material; however sometimes they are affected by the activators or inhibitors present in the material being standardised.

These inexpensive methods are used for analysing large batches of samples, but 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 (figure).

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 4mm 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.