Microbiological assays accuracy

Microbiological assay should stress accuracy over precision. Standardization of an assay method should include comparisons with at least one other organism having a different nutritional pattern and specificity toward the compound being assayed. Such a comparison was made for cyanocobalamin (vitamin Bi2 ) content of human blood and serum, using four microorganisms differing in their cobamide requirements and metabolism (B9). 

It is an essential condition of biological assay methods that the tests on the standard preparation and on the sample whose potency is being determined should be carried out at the same time and, in all other respects, under strictly comparable conditions. The validation of microbiological assay method includes performance criteria (analytical parameters) such as linearity, range, accuracy, precision, specificity, etc. 

Research into new analytical techniques for foodstuffs continues, striving for greater accuracy, sensitivity or simplicity, for more rapid methods, for simultaneous multielement analysis, etc. Chromatographic techniques, e.g., LC, GLC, GC-MS, have led to great improvements in the levels of accuracy, sensitivity, and detection that can be achieved for many analytes including carbohydrates, certain vitamins, chemical residues, and additives. Work is still required, for instance, in the area of vitamin analysis in order to provide standard techniques that are applicable to all food types and that would enable concurrent multi-vitamin analysis to take place. Many of the microbiological assays currently used for vitamin determination involve long incubation times and more rapid techniques are needed. 

There are various microbiological, chemical and animal assays available for thiamine analysis. Method selection depends on the accuracy and sensitivity required, and on the interferences due to the sample matrix. Although microbiological assays do exist for thiamine, these are rarely used. The highly fluorescent products are the basis of the thiochrome procedure for thiamine quantiflcation. Thiochrome analysis has been used to develop most of the analytical data available on the thiamine content of the food supply. 

Any transformation that could be affected for such curves so that they would yield linear graphs would have manifest advantages, because it would afford a sound mathematical basis for computation, which is a desirable consideration for any analytical method. In any type of microbiological assay of vitamin Bu the results should be plotted to test whether a dose-response, a log dose-response, or a log dose-log response curve is sufficiently linear to make possible the use of statistical methods. In the assay of vitamin B12 activity in crude materials, however, the most pertinent problem at present is to relate this activity to certain defined substances rather than to obtain the highest accuracy in measuring the Bu activity. 

In more recent times chemically defined basal media have been elaborated, on which the growth of various lactic acid bacteria is luxuriant and acid production is near-optimal. The proportions of the nutrients in the basal media have been determined which induce maximum sensitivity of the organisms for the test substance and minimize the stimulatory or inhibitory action of other nutrilites introduced with the test sample. Assay conditions have been provided which permit the attainment of satisfactory precision and accuracy in the determination of amino acids. Experimental techniques have been provided which facilitate the microbiological determination of amino acids. On the whole, microbiological procedures now available for the determination of all the amino acids except hydroxy-proline are convenient, reasonably accurate, and applicable to the assay of purified proteins, food, blood, urine, plant products, and other types of biological materials. On the other hand, it is improbable that any microbiological procedure approaches perfection and it is to be expected that old methods will be improved and new ones proposed by the many investigators interested in this problem. 

The fundamental parameters for bioanalytical validations include accuracy, precision, selectivity, sensitivity, reproducibility, stability of the drug in the matrix under study storage conditions, range, recovery, and response function (see Section 8.2.1). These parameters are also applicable to microbiological and ligand-binding assays. However, these assays possess some unique characteristics that should be considered during method validation, such as selectivity and quantification issues. 

Recent advances have enormously widened the range of applicability of amino acid determinations as a control on fractionation. Not only have colorimetric procedures been revised and increased in accuracy, but the development of microbiological and chromotographic methods of assay now permits the quantitative determination of almost any amino acid residue, using very small amounts of protein of the order of 10 to 100 mg. The results are rapid and, by some at least of tiiese methods, appear to be trustworthy. Since the general principles of all these methods have been reviewed in the previous volume of this series, by Martin and Synge (142) and by Snell (197), no further discussion of them need be given here. 

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