Microbiological assay is applicable only to the B vitamins. The rate of growth of a species of microorganism that requires a vitamin is measured in growth media that contain various known quantities of a foodstuff preparation containing unknown amounts of the vitamin. The response (measured as rate of growth) to the unknown amounts of vitamin is compared with that obtained from a known quantity of the pure vitamin. Depending on the way in which the food sample was prepared, the procedure may indicate the availability of the vitamin in the food sample to the microorganism.

Animal assay

All of the vitamins, with the exception of vitamin B₁₂, can be estimated by the animal-assay technique. One advantage of this method is that animals respond only to the biologically active forms of the vitamins. On the other hand, many other interfering and complicating factors may arise; therefore, experiments must be rigidly standardized and controlled. Simultaneous estimates usually are made using a pure standard vitamin preparation as a reference and the unknown food whose vitamin content is being sought; each test is repeated using two or more different amounts of both standard and unknown in the assays listed below.

In a growth assay, the rat, chick, dog (used specifically for niacin), and guinea pig (used specifically for vitamin C) usually are used. One criterion used in a vitamin assay is increase in body weight in response to different amounts of a specific vitamin in the diet. There are two types of growth assay. In a prophylactic growth assay, the increase in weight of young animals given different amounts of the vitamin is measured. In a curative growth assay, weight increase is measured in animals first deprived of a vitamin and then given various quantities of it. The curative growth assay tends to provide more consistent results than the prophylactic technique.

In a reaction time assay, an animal is first deprived of a vitamin until a specific deficiency symptom appears; then the animal is given a known amount of a food extract containing the vitamin, and the deficiency symptom disappears within a day or two. The time required for the reappearance of the specific symptoms when the animal again is deprived of the vitamin provides a measure of the amount of vitamin given originally. The graded response assay, which may be prophylactic or curative, depends on a characteristic response that varies in degree with the vitamin dosage. An example of this technique is an assay for vitamin D in which the measured ash content of a leg bone of a rat or chick is used to reflect the amount of bone calcification that occurred as a result of administration of a specific amount of vitamin D. In an all-or-none assay, the degree of response cannot be measured; an arbitrary level is selected to separate positive responses from negative ones. The percent of positively reacting animals provides a measure of response; i.e., vitamin E can be measured by obtaining the percent of fertility in successfully mated female rats.