Theory and Method
The Guthrie test is a semiquantitative assay designed to detect elevated blood levels of the amino acid phenylalanine, using the ability of phenylalanine to facilitate bacterial growth in a culture medium with an inhibitor.
A drop of blood is usually obtained by pricking the heel of a newborn infant in a hospital nursery six or seven days after birth (end of the first week). The blood is collected on a piece of filter paper and sent to a central laboratory. A small disk of the filter paper is punched out and placed on an agar gel plate containing Bacillus subtilis and B-2-thienylalanine. Each gel holds 60-80 disks. The agar gel is able to support bacterial growth but the B-2-thienylalanine inhibits bacterial growth. However, in the presence of extra phenylalanine leached from the impregnated filter paper disk, the inhibition is overcome and the bacteria grow. Within a day the bacterial growth surrounding the paper disk is visible. The amount of growth, measured as the diameter of the colony, is roughly proportional to the amount of phenylalanine in the serum. The result is read by comparing the diameter of each sample disk's colony to the colonies of a series of reference disks with standard phenylalanine content included on each large plate.
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