Biomedical subjects
Publications and source records attributed to F Kavanagh.
Computational procedures specified for the FDA single-dose diffusion assay for antibiotics may cause substantial error in estimated sample potency. An unrecognized mistake in reference solution concentration is the source of error. It is caused by correcting responses from standard and sample plates differently. The error can be avoided by correcting both standard and sample responses to the observed reference response.
Factors that may influence the accuracy and precision of microbiological manual and semiautomated turbidimetric methods as well as diffusion assays are discussed. Influence of kind of equipment, media, test bacteria, sample preparation, form of dose response lines, operations, and personnel on quality of assays is examined with the objective of reducing to insignificance those factors under control of the analyst that are responsible for low quality assays.
Procedures were devised to linearize the usually curved calibration lines for turbidimetric microbiological assays. Three new equations relating concentration of drug and turbidity are described; two are for antibiotic assays and one for vitamin assays. One equation is for antibiotic assays employing Klebsiella pneumoniae as the test organism. The accuracy of interpolation from the three equations was studied by means of appropriate mathematical models based on erthromycin, chlortetracycline (K. pneumoniae), and cyanocobalamin assays. The accuracy of the new expressions was significantly superior to those used previously, and they are of general applicability.
The accuracy of an automated system for the microbiological assay of antibiotics was increased by improvement attendant to connection to an on-line computer. The system was used to investigate the suitability of four forms of interpolation formulas by assaying for chlortetracycline and erythromycin. The calibration lines were prepared as point-to-point straight-line approximations and as cubic equations. Cubic equations through four calibration points were preferred. Since the automated system was a four-channel instrument, a separate response line was prepared for each channel. Combining the four response lines into one could substantially degrade the accuracy and precision of assays. A new general equation relating the response of the test organism to concentrations of active materials was used to account for factors in addition to the antibiotic upon the dose-response line. Some of these factors were: diluents, growth substances, relative proportions of mixed antibiotics, pH and buffer capacities of the sample solution and assay broth, salts, and organic compounds in samples and not in standard solutions. The equation was used to show under what conditions the dose-response lines of mixtures and single-component antibiotics could be the same. It could also account for the nonspecific nature of turbidimetric assays. The equation showed assay biases to be caused not by differences in composition of antibiotics in standards and samples but by differences in other substances affecting growth of the test organism. A new dose-response line applicable to assays using Klebsiella pneumoniae was described.
Application of new equipment and new techniques was made to antibiotic diffusion assays. Accumulation of data and computation of potencies were made by an on-line computer. The system was tested by assaying cephalexin with the aid of Bacillus subtilis in an FDA single-dose design modified by reducing the number of standards to two. The influence of the thickness of the base layer and the form of the dose-response line were tested. Zone diameter was measured with a resolution of 0.01 mm. The potency of samples was measured with an error usually less than 3%. An error of 0.1 mm in measuring zone size would cause an error of 3% of potency. The usual calibration line was inadequate for extrapolation beyond a twofold range. A dose-response line derived from the Cooper equation was better for standard curves spanning more than a twofold range of concentrations. Precision was better on the plates with the 20-ml base layer. The two-dose method of assaying gave larger errors than the single-dose method. Large variances in measuring zone diameters are a reason for the low precision of diffusion assays and set an inherent limit on precision.
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